Hybridization-triggered fluorescent detection of nucleic acids

ABSTRACT

Compositions and methods for fluorescent detection of nucleic acids are provided. The compositions can be detected by fluorescence when hybridized to a nucleic acid containing a target sequence, but are non-fluorescent in the non-hybridized state. Alternatively, the fluorescence properties of the compositions change in a detectable manner upon hybridization to a nucleic acid containing a target sequence. Methods for synthesis and methods of use of the compositions are also provided.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/227,001, filed Aug. 21, 2002, now U.S. Pat. No. 6,951,930 which is adivisional application of U.S. application Ser. No. 09/428,236, filedOct. 26, 1999, now U.S. Pat. No. 6,472,153.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present invention is in the field of molecular biology. Morespecifically, the invention is in the field of assays that utilizefluorescently-labeled probes and primers in hybridization assays fordetection of nucleic acids.

BACKGROUND

The use of fluorescent molecules in the biological sciences for researchand diagnostic purposes is well known. See, for example, Kirkbright“Fluorescent Indicators” in Indicators, (ed. Bishop, E.) Pergamon Press,New York, Chapter 9, pp. 685-708, 1972; and Haugland (1996) Handbook ofFluorescent Probes and Research Chemicals, Sixth edition, MolecularProbes, Inc., Eugene, Oreg. Fluorescent moieties have been used fornon-specific labeling of single- and double-stranded nucleic acids(e.g., acridine, ethidium bromide) and for labeling of nucleic acidprobes that are used in sequence-specific detection of nucleic acidtargets. In general, when fluorescent nucleic acid binding moleculesand/or fluorescently-labeled probes are used for nucleic acid detection,unbound fluorescent material must be removed from the system, prior toanalysis, to maximize detection of a signal. If unbound material is notremoved, background fluorescence leads to a reduction in thesignal:noise ratio.

Compositions which are fluorescent when bound to double-stranded DNA,but which do not fluoresce (or fluoresce at a different wavelength) whenunbound, have been described. See, for example, Haugland, supra, pp.144-156 and 161-174, especially pp. 161-165. Although such compositionsmay exhibit fairly general sequence preferences (e.g., for AT-rich vs.GC-rich target sequences), they are not capable of eithersequence-specific detection of a target or of mismatch discriminationbetween targets having related but non-identical sequences. In addition,such compositions cannot be used for multiplex detection of targetsequences (i.e., simultaneous detection of more than one targetsequence).

Several new analytical techniques depend on sequence-specific detectionand mismatch discrimination using fluorescence as a readout. Forinstance, homogeneous detection methods for monitoring the accumulationof specific PCR products have recently been developed. One of theseassays utilizes an oligonucleotide probe which contains a fluorescentmolecule at its 5′ end and a fluorescence quencher at its 3′ end.Because of the presence of the quencher, the oligonucleotide probe doesnot exhibit fluorescence, or exhibits relatively low fluorescence, inthe single-stranded state. The assay exploits the 5′→3′ nucleaseactivity of Taq DNA polymerase to hydrolyze such a probe after it hasformed a sequence-specific duplex with a target nucleic acid. Hydrolysisreleases the fluorescent molecule from the 5′ end of the probe, removingit from proximity with the quencher, thereby allowing increasedfluorescence to occur. Lee et al. (1993) Nucleic Acids Res.16:3761-3766. In another recently-developed technique, microvolumemulti-sample fluorimeters with rapid temperature control have beendeveloped for use with 5′-nuclease assays using double-labeledfluorescent probes. Wittwer et al. (1997) Biotechniques 22:176-181. U.S.Pat. No. 5,871,908 describes a homogeneous assay in which fluorescentsignal varies with a temperature gradient and the variation is detectedin real time. However, all of these assays involve post-hybridizationdetection steps, often involving the use of enzymes, which are costly,time-consuming and can be difficult to regulate, in terms of theiractivity.

There is thus a need for sensitive and straightforward methods andcompositions for sequence-specific detection of nucleic acid targets; inparticular fluorescent detection. Besides the advantages of usingfluorescent molecules as an alternative to radioisotopes, improvementsin speed, economy and convenience would attend the development of amethod in which the hybridization event itself provided a directreadout, without requiring subsequent detection steps, such as enzymatictreatment of hybridized material.

Tyagi et al. (1996) Nature Biotechnol 14:303-308 described probescontaining a fluorophore and a quencher molecule which, in theunhybridized state, form a hairpin which brings the fluorophore and thequencher into proximity so that fluorescence is quenched. Uponhybridization, the hairpin structure is disrupted and fluorescence isobserved. Such probes require the attachment of both a fluorophore and aquencher, and also must contain regions of self-complementarity, whichmay interfere with their ability to hybridize to their target.

Minor groove binding agents that non-covalently bind within the minorgroove of double stranded DNA have been described. Zimmer et al. (1986)Prog. Biophys. Molec. Biol. 47:31-112; Levina et al. (1996) Antisense &Nucl. Acid Drug Develop. 6:75-85. Hybridization assays using anoligonucleotide coupled to a minor groove binder (MGB) have beendescribed in U.S. Pat. No. 5,801,155, and in International PatentApplication No. PCT/US99/07487. These publications describe the abilityof minor groove binders, when conjugated to an oligonucleotide, toincrease the ability of the oligonucleotide to distinguish between aperfectly-matched target sequence and a target sequence with asingle-nucleotide mismatch. This heightened discriminatory ability ofMGB-oligonucleotide conjugates is reflected in a greater difference inmelting temperature (T_(m)) between matched and mismatched duplexesformed with an MGB-oligonucleotide conjugate, on the one hand, andmatched and mismatched duplexes formed with an unmodifiedoligonucleotide, on the other. The aforementioned U.S. Pat. No.5,801,155, and International Patent Application No. PCT/US99/07487additionally disclose that a duplex comprising a MGB-oligonucleotideconjugate has a higher melting temperature than a duplex of identicalsequence comprising an unmodified oligonucleotide. This property ofduplexes comprising a MGB-oligonucleotide conjugate allows more faciledetection of related mismatched sequences with a MGB-oligonucleotideprobe, and enables the use of shorter oligonucleotide probes in PCRamplification reactions, if the probe is conjugated to a MGB. Thesepublications also describe the use of an oligonucleotide coupled to aminor groove binder, a fluorophore and a fluorescent quencher, inhydrolyzable probe assays.

Intercalating agents are, generally speaking, flat aromatic moleculesthat bind non-covalently to double-stranded DNA or RNA by positioningthemselves between adjacent base pairs of the duplex. Gago (1998) Method14:277-292. U.S. Pat. No. 4,835,283 and PCT publication WO 98/50541, forexample, disclose oligonucleotides that are covalently bound to anintercalating group. Oligonucleotides conjugated to either minor groovebinders or intercalating groups can be used in hybridization assays.

Hoechst 33258 and 33342 are examples of fluorescent dyes that bind inthe minor groove of DNA duplexes. A conjugate consisting of anoligonucleotide coupled to a Hoechst-like minor groove binder has beenobserved to show increased fluorescence upon hybridization to asingle-stranded target. O'Donnell et al. (1995) Biorg. Med. Chem.3:743-750; and Wiederholt et al. (1996) J. Amer. Chem. Soc.118:7055-7062. This conjugate consisted solely of an oligonucleotidebound to a MGB.

EP 231 495 discloses a polynucleotide compound comprising at least twoentities, which upon hybridization is capable of generating a change inproperty of the hybrid.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for improvedhybridization detection and mismatch discrimination by fluorescence. Inthe practice of the invention, an increase in fluorescent signal, achange in fluorescence excitation and/or emission, and/or some otherchange in fluorescence properties occurs after hybridization of anoligonucleotide, appropriately labeled with a latent fluorophore and aminor groove binder, to a complementary target.

In one aspect, the present invention encompasses a covalently boundoligonucleotide (ODN)/minor groove binder (MGB)/latent fluorophore (LF)combination. The oligonucleotide comprises a plurality of nucleotides(and/or modified nucleotides and/or nucleotide analogues), a 3′ end anda 5′ end. A minor groove binder moiety is a radical of a molecule havinga molecular weight of approximately 150 to approximately 5000 Daltonswhich molecule binds in a non-intercalating manner into the minor grooveof non-single-stranded nucleic acids or hybrids, analogues and chimerasthereof (i.e., double- or triple-stranded polynucleotides) with anassociation constant greater than approximately 10³M⁻¹. The minor groovebinder moiety is covalently attached at the 3′ end and/or the 5′ end,and/or to at least one of said nucleotides, modified nucleotides and/ornucleotide analogues of the oligonucleotide, and is typically attachedto the oligonucleotide through a first linking group having a backbonelength of no more than about 100 atoms. A latent fluorophore is aradical of a molecule having a molecular weight of approximately 150 toapproximately 5000 Daltons which binds in an intercalating manner intonon-single-stranded nucleic acids or hybrids, analogues and chimerasthereof, or lies preferentially in the minor groove, or in anothermanner is oriented to the DNA molecule by the minor groove binder moietyso that it becomes fluorescent or its fluorescence properties arechanged in a detectable way. Typically, the latent fluorophore isattached to the minor groove binder moiety through a second linkinggroup having a backbone length of no more than about 50 atoms.

In one embodiment, the ODN-MGB-LF conjugate is relativelynon-fluorescent in its single-stranded state, but becomes fluorescentafter hybridization to a target sequence. In another embodiment, theODN-MGB-LF conjugate may exhibit some fluorescence emission at one ormore particular wavelengths in its single-stranded state, but, afterhybridization, its maximal fluorescence emission is shifted to adifferent wavelength. In yet another embodiment, the wavelength at whichmaximal fluorescence excitation occurs can change after hybridization ofan ODN-MGB-LF conjugate.

In another aspect, the present invention encompasses processes for thesynthesis of covalently-bound oligonucleotide-minor groove binder-latentfluorophore conjugates. The invention also provides novel compositionsfor use in the synthesis of ODN-MGB-LF conjugates.

In yet another aspect, the invention relates to the use of compositionscomprising an oligonucleotide, a minor groove binder and a latentfluorophore, in covalent or functional linkage, as hybridization probesfor fluorescent detection in analytical and diagnostic methods. Thesemethods include but are not limited to, PCR (including real-time PCR),single nucleotide mismatch discrimination, target amplification, signalamplification and assays utilizing oligonucleotide arrays.

In an exemplary method for detecting a target sequence in apolynucleotide, an ODN-MGB-LF conjugate is combined with a samplecontaining a polynucleotide to form a hybridization mixture, wherein theODN portion of the conjugate comprises a sequence which hybridizes tothe target sequence, the hybridization mixture is incubated underconditions which yield specific hybridization, and thereafterfluorescence of the hybridization mixture is measured, whereinfluorescence is indicative of the presence of the target sequence.

In another embodiment, the compositions and methods of the invention areused for detection of a target sequence in a polynucleotide, wherein thepolynucleotide is in a sample comprising a plurality of polynucleotideshaving different sequences.

In yet another embodiment, the compositions and methods of the inventionare used for detection of a target sequence in a polynucleotide, whereinthe polynucleotide is present in a mixture of other polynucleotides, andwherein one or more of the other polynucleotides in the mixture comprisesequences that are related but not identical to the target sequence. Inthis embodiment, an ODN-MGB-LF conjugate is contacted with theaforementioned mixture of polynucleotides, wherein the ODN-MGB-LF formsa stable hybrid only with a target sequence that is perfectlycomplementary to the oligonucleotide portion of the composition andwherein the composition does not form a stable hybrid with any of therelated sequences. After hybridization, the fluorescence of the mixtureis measured, wherein fluorescence is indicative of the presence of thetarget sequence.

In a further embodiment, the compositions and methods of the inventionare used for single-nucleotide mismatch discrimination.

In one embodiment, the compositions are used for the detection ofsingle-stranded nucleic acids. The ODN portion of the ODN-MGB-LFconjugate forms a duplex with a single-stranded target nucleic acid, andinteractions of the MGB and LF portions of the conjugate with theresulting duplex nucleic acid result in enhanced fluorescence, or someother change in the fluorescence properties of the latent fluorophore.

In another embodiment, the compositions of the invention are used fordetection of double-stranded nucleic acid targets. In this case the ODNportion of the conjugate is a triplex-forming oligonucleotide. See, forexample, Fresco, U.S. Pat. No. 5,422,251; Hogan, U.S. Pat. No.5,176,996; and Lampe (1997) Nucleic Acids Res. 25:4123-4131. Formationof a triplex between the conjugate and a double-stranded target resultsin enhanced fluorescence, or some other change in the fluorescenceproperties of the latent fluorophore.

In another embodiment, the invention provides compositions and methodsfor the simultaneous detection of multiple target sequences in a sample(i.e., multiplex detection).

In another embodiment, the invention provides compositions and methodsfor amplification of a target sequence, wherein the amplificationprimer(s) are capable of hybridization-triggered fluorescence. Thisembodiment is particularly suitable for various amplification methods inwhich the product is detectable in real time.

In further aspects, ODN-MGB-LF conjugates are immobilized on a solidsupport, preferably in an ordered array. An immobilized conjugate can beused for capture of a target polynucleotide and/or as a primer using acaptured polynucleotide as a template. In these and other applications,the compositions of the invention are able to discriminate betweenclosely related polynucleotide sequences.

In another aspect, the invention provides kits for fluorescent detectionof nucleic acids, and for mismatch discrimination between relatednucleic acids, wherein the kits comprise at least one ODN-MGB-LFconjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representations of ground (S_(o)) and excited singlet(S_(l)) states for an exemplary cyanine dye. FIG. 1A depictsradiationless decay by free dye. FIG. 1B shows fluorescent emission whenrotation about the cyanine-methine bond is restricted, for example, byintercalation of the dye into a nucleic acid.

FIG. 2. Fluorescence of single- and double-stranded thiazoleorange-minor groove binder-oligonucleotide conjugates. FIG. 2A showshybridization-triggered fluorescence with a conjugate,TO-MGB-5′-CAATTTAAAGAA-3′ (SEQ ID NO: 1), containing an AT-richsequence; FIG. 2B shows hybridization-triggered fluorescence with aconjugate, TO-MGB-5′-TTCCCGAGCGGC-3′ (SEQ ID NO: 2), containing aGC-rich sequence. See Example 1, infra, for hybridization conditions.

FIG. 3. Effect of a minor groove binder on hybridization-triggeredfluorescence. FIG. 3A shows fluorescence of the ODN-MGB-LF conjugateTO-MGB-5′-CAATTTAAAGAAAAGAAG-3′ (SEQ ID NO: 3), as a function of itsconcentration, in the presence of an equimolar concentration of itscomplementary sequence. FIG. 3B shows fluorescence of a ODN-TOconjugate, containing the same sequence but lacking a MGB, as a functionof its concentration in the presence of an equimolar concentration ofits complementary sequence. “F” on the ordinate refers to fluorescenceintensity, in arbitrary units. See Example 1, infra, for hybridizationconditions.

FIG. 4. Hybridization-triggered fluorescence in a DNA-RNA hybrid. FIG.4A shows fluorescence spectra of a 15-mer polydT-MGB-(2-dimethylaminonaphthalene-6-sulfonamide) conjugate (SEQ IDNO:24) at a concentration of 1×10⁻⁷ M (lower trace, labeled “SS probe”)and a hybrid of this probe with a two-fold molar excess of a poly rAtarget (upper trace, labeled “Hybrid duplex”). Hybridization wasconducted in 10 mM phosphate, 0.15 M NaCl, 1 mM EDTA, pH 7.4 for 15 mmat 25° C. FIG. 4B shows the structure of the conjugate (SEQ ID NO:20).

FIG. 5. Discrimination between matched and mismatched target sequences.Fluorescence of conjugate 3 (see Table 2), at a concentration of6.7×10⁻⁷ M, was measured as a function of the concentration of itstarget sequence. In the upper curve (solid circles), the target wasperfectly complementary to the ODN portion of the conjugate, having thesequence 5′-TTTCTTAAAACGAATTT-3′ (SEQ ID NO: 4). In the lower curve, thetarget, 5′-TTTCTTAACACGAATTT-3′ (SEQ ID NO: 5), had a single-nucleotidemismatch with respect to the ODN portion of the conjugate, as indicatedby underlining. Hybridization was conducted in pH 7.4 buffer (10 mMphosphate, 0.15 M NaCl, 1 mM EDTA) at 25 C for 15 min.

FIG. 6. Single-nucleotide mismatch discrimination by real-time PCR usingODN-MGB-TO conjugates as primers. Symbols are as follows: diamonds:matched primer-TO conjugate (no MGB); squares: matched primer-MGB-TOconjugate; triangles: mismatched primer-MGB-TO conjugate; X: matchedprimer-MGB-TO conjugate, no template. See Example 9 for details.

MODES FOR CARRYING OUT THE INVENTION

This invention is directed to the concept of hybridization-triggeredfluorescence detection of nucleic acids and provides the basis for a newclass of diagnostic probes for detection and mismatch discrimination ofspecific DNA and/or RNA sequences.

The basic constructs of the invention involve covalent conjugates of anoligonucleotide, a minor groove binder and a potentially fluorogenicreporter group. In one configuration, conjugates of the invention havethe structure ODN-MGB-LF. These can constitute an essentially lineararrangement of the ODN, MGB and LF components such that a MGB has an ODNattached to one end and a LF to the other, or an arrangement in which anODN and a LF are attached to the same end of a MGB. In anotherconfiguration, the conjugates of the invention have a fluorogenicreporter group covalently interposed between an oligonucleotide and aminor groove binder, to give a structure which can be representedODN-LF-MGB.

The fluorogenic reporter group is chosen such that hybridization of theoligonucleotide to a complementary target sequence results in anenhancement, at a particular wavelength, in the fluorescence quantumyield of the fluorogenic reporter group. Accordingly, the fluorogenicreporter group is also known as a latent fluorophore (LF). Enhancementin fluorescence intensity can result from binding of the reporter groupto the hybrid formed between the oligonucleotide and the targetsequence, from a particular positioning of the reporter group withrespect to the hybrid thus changing the environment of the fluorogenicreporter, from intercalation of the reporter group into the hybrid,and/or from restriction of rotational movement of the fluorogeniccompound as a result of hybridization.

For the purposes of the invention, hybridization includes interaction ofan oligonucleotide with a single-stranded nucleic acid to form a duplex,as well as interaction of an oligonucleotide with a double-strandednucleic acid to form a triplex. For detection of double-stranded nucleicacid targets, the oligonucleotide portion of the composition is atriplex-forming oligonucleotide. Design of triplex formingoligonucleotides, based on non-Watson-Crick base-pairing schemes, suchas Hoogsteen and reverse Hoogsteen base pairing, is well-known to thoseof skill in the art. See, for example, Fresco, supra; Hogan, supra;Lampe, supra; and Ornstein et al. (1983) Proc. Natl. Acad. Sci. USA80:5171-5175. For detection of a duplex target, a triplex-formingoligonucleotide is linked to a MGB through an appropriate linker havinga backbone of approximately 100 atoms (Kutyavin et al (1997) NucleicAcids Res. 25:3718-3723), and the MGB is in turn linked to a latentfluorophore through a linker of approximately 50 atoms preferebly 40atoms, more preferably 30 atoms, more preferably 20 atoms, still morepreferably 10 atoms and most preferably 5-6 atoms.

The invention provides selected latent fluorophore-MGB-oligonucleotideconjugates which exhibit increased fluorescence upon hybridization,compared to the latent fluorophore-MGB-oligonucleotide conjugate alone.The invention thus combines the enhanced hybrid stability and mismatchdiscrimination obtained with MGB-oligonucleotide conjugates (see, forexample, U.S. Pat. No. 5,801,155, and International Patent ApplicationNo. PCT/US99/07487) with the speed, simplicity and sensitivity ofdetection by hybridization-triggered fluorescence.

The practice of the invention will employ, unless otherwise indicated,conventional techniques in organic chemistry, biochemistry,oligonucleotide synthesis and modification, bioconjugate chemistry,nucleic acid hybridization, molecular biology, microbiology, genetics,recombinant DNA, and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Maniatis, Fritsch & Sambrook, MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory Press (1982); Sambrook, Fritsch &Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, ColdSpring Harbor Laboratory Press (1989); Ausubel, et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (1987 and annualupdates); Gait (ed.), OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH,IRL Press (1984); Eckstein (ed.), OLIGONUCLEOTIDES AND ANALOGUES: APRACTICAL APPROACH, IRL Press (1991).

The disclosures of all publications and patents cited herein are herebyincorporated by reference in their entirety.

Oligonucleotides

Broadly speaking, the oligonucleotide portion of an ODN-MGB-LF conjugatecomprises approximately 3 to 100 nucleotide units. However, longeroligonucleotides are also encompassed by the invention, and the termoligonucleotide is not intended to be limiting with respect to thelength of the molecule to which the term refers. The nucleotide unitswhich are incorporated into the ODNs in accordance with the presentinvention include the major heterocyclic bases naturally found innucleic acids (uracil, cytosine, thymine, adenine and guanine) as wellas naturally-occurring and synthetic modifications and analogues ofthese bases such as, for example, hypoxanthine, 2-aminoadenine,2-thiouracil, 2-thiothymine, 5-N⁴ ethenocytosine,4-aminopyrrazolo[3,4-d]pyrimidine and6-amino-4-hydroxy-[3,4-d]pyrimidine. Any modified nucleotide ornucleotide analogue compatible with hybridization of the ODN-MGB-LFconjugate to a target sequence is useful in the practice of theinvention, even if the modified nucleotide or nucleotide analogue itselfdoes not participate in base-pairing, or has altered base-pairingproperties compared to naturally-occurring nucleotides.

The sugar or glycoside portion of the ODN portion of the conjugates cancomprise deoxyribose, ribose, 2-fluororibose, and/or 2-O-alkyl oralkenylribose wherein the alkyl group comprises 1 to 6 carbon atoms andthe alkenyl group comprises 2 to 6 carbon atoms. In thenaturally-occurring nucleotides, modified nucleotides and nucleotideanalogues that can comprise an ODN, the sugar moiety forms a furanosering, the glycosidic linkage is of the β configuration, the purine basesare attached to the sugar moiety via the purine 9-position, thepyrimidines via the pyrimidine 1-position and the pyrazolopyrimidinesvia the pyrazolopyrimidine 1-position (which is equivalent to the purine9-position). In a preferred embodiment, the sugar moiety is2-deoxyribose; however, any sugar moiety known to those of skill in theart, that is compatible with the ability of the oligonucleotide portionof the compositions of the invention to hybridize to a target sequence,can be used.

In one embodiment, the nucleoside units of the ODN portion of theconjugate are linked by a phosphodiester backbone, as is well known inthe art. In additional embodiments, internucleoside linkages can includeany linkage known to one of skill in the art that is compatible withspecific hybridization of the ODN including, but not limited tophosphorothioate, methylphosphonate, sulfamate (e.g., U.S. Pat. No.5,470,967) and polyamide (i.e., peptide nucleic acids). Peptide nucleicacids are described in Nielsen et al. (1991) Science 254: 1497-1500;U.S. Pat. No. 5,714,331; and Nielsen (1999) Curr. Opin. Biotechnol.10:71-75. Thus, for example, part or all of the ODN portion of theconjugate can be a peptide (polyamide) nucleic acid (PNA).

In certain embodiments, the ODN portion of the conjugate can be achimeric molecule; i.e., the ODN can comprise more than one type of baseor sugar subunit, and/or the linkages can be of more than one typewithin the same ODN. For example, the ODN can be a PNA/DNA chimera. See,for example, Nielsen (1999) supra; and Koch et al. (1995) TetrahedronLetts. 36:6933-6936. In addition, the ODN can be interrupted bynon-nucleotide components.

The ODN portion of the ODN-MGB-LF conjugates can comprise a tail moietyattached at either the 3′ or 5′-end. The tail moiety is distinguishedfrom the minor groove binding moiety, which is preferably also attachedto the 3′ or 5′ end of the ODN, or to both. The tail moiety, if present,is attached to the end of the ODN which does not bear the minor groovebinder moiety. By way of example, a tail moiety can be a phosphate, aphosphate ester, an alkyl group, an aminoalkyl group, a lipophilicgroup, or a molecule as disclosed, for example, in U.S. Pat. Nos.5,512,667; 5,419,966; 5,574,142 and 5,646,126.

Variations of the bases, sugars, internucleoside backbone and tailmoieties of the ODN portion of ODN-MGB-LF conjugates will be compatiblewith the ability of the conjugates to bind to a target sequence in amanner in which the minor groove binding moiety is incorporated in thenewly formed duplex or triplex and thereby increases the meltingtemperature of the newly formed duplex, (i.e., increases the stabilityof the hybrid) as described in U.S. Pat. No. 5,801,155; InternationalPatent Application No. PCT/US99/07487; Kutyavin et al., supra and Kamuret al. (1998) Nucleic Acids Res. 26:831-838; and with the ability of theLF to undergo hybridization-triggered fluorescence.

Formation of a hybrid between an ODN-MGB-LF conjugate and a targetsequence results in an increase in fluorescence quantum yield or achange in the absorption and/or emission spectra of the LF. In light ofthe foregoing, those skilled in the art will readily understand that theprimary structural limitation of the various component parts of the ODNportion of the ODN-MGB-LF conjugate are related to the ability of theODN portion to form a hybrid with a specific target sequence. Thus, alarge number of structural modifications, both known and to bedeveloped, are possible within these bounds. Moreover, synthetic methodsfor preparing the various heterocyclic bases, sugars, nucleosides andnucleotides which form the ODN portion of ODN-MGB-LF conjugates arewell-developed and known in the art. For example,N₄,N₄-ethano-5-methyldeoxycytidine, its nucleoside, nucleotide and/oroligonucleotides incorporating this base are synthesized in accordancewith the teachings of Webb et al. (1986) Nucleic Acids Res.,14:7661-7674; and Webb et al. (1986) J. Am. Chem. Soc. 108:2764.4-aminopyrazolo[3,4-d]pyrimidine,6-amino-4-hydroxypyrazolo[3,4-d]pyrimidine, their nucleosides,nucleotides and oligonucleotides incorporating these bases aresynthesized in accordance with the teachings of Kazimierczuk et al.(1984) J. Am. Chem. Soc. 106:6379-6382. Preparation of oligonucleotidesof specific predetermined sequence is conducted in accordance with thestate of the art. A preferred method of oligonucleotide synthesisincorporates the teaching of U.S. Pat. No. 5,419,966.

Minor Groove Binders

In duplex DNA, the two antiparallel phosphodiester backbones do not liedirectly opposite each other across the longitudinal axis of the duplexmolecule; rather they are offset. As a result, the surface of the duplexcontains two differently-sized grooves: a major groove and a minorgroove. The minor groove lies between the 1′ C. atoms of the sugars onopposite strands, forming a cleft with a width of 5.7 Å, and a depth of7.5 Å, which pursues a helical path along the surface of the duplex.Minor groove binders are molecules that, by virtue of their size and/orstructure, are capable of interacting with this structural feature ofduplex and triplex polynucleotides.

As noted supra, a minor groove binder (MGB) is a molecule that bindswithin the minor groove of double stranded nucleic acid, including DNA,RNA, DNA-RNA hybrids and nucleic acid chimeras, such as PNA/DNAchimeras. Minor groove binders have widely varying chemical structures,all of which are capable of binding within a minor groove having thegeometry and dimensions described above. For example, certain MGBs arecapable of forming a crescent-shaped three dimensional structure. Manyminor groove binding compounds have a strong preference for A+T (adenineand thymine)-rich regions of the B form of double-stranded DNA. Withoutwishing to be bound by theory, it is possible that this preference isdue, at least in part, to steric interference of MGB binding by the2-amino group of guanine. However, if guanine is replaced byhypoxanthine in an ODN-MGB-LF conjugate, the potential for stericinterference is reduced and binding of a MGB conjugate to a G+C-richsequences is enhanced. Accordingly, ODN-MGB-LF conjugates incorporatinga radical or moiety derived from a minor groove binder molecule havingpreference for both A+T-rich and G+C-rich regions are within the scopeof the invention.

Examples of minor groove binding compounds which can, in accordance withthe present invention, be covalently bound to ODNs to form the novelODN-MGB-LF conjugates include certain naturally-occurring compounds suchas netropsin, distamycin, lexitropsin, mithramycin, chromomycin A₃,olivomycin, anthramycin, and sibiromycin, as well as related antibioticsand synthetic derivatives. Certain bisquartemary ammonium heterocycliccompounds, diarylamidines such as pentamidine, stilbamidine and berenil,CC-1065 and related pyrroloindole and indole polypeptides, Hoechst33258, 4′-6-diamidino-2-phenylindole (DAPI), and as a number ofoligopeptides consisting of naturally-occurring or synthetic amino acidsare minor groove binder compounds. The chemical structures of severalexemplary MGBs are illustrated below.

For the purposes of the invention, a molecule is a MGB if it is capableof binding within the minor groove of double-stranded DNA,double-stranded RNA, DNA-RNA hybrids, DNA-PNA hybrids, hybrids in whichone strand is a PNA/DNA chimera and/or polymers containing purine and/orpyrimidine bases and/or their analogues which are capable ofbase-pairing to form duplex, triplex or higher order structurescomprising a minor groove, wherein said binding occurs with anassociation constant of 10³ M⁻¹ or greater. Such binding can be detectedby any method known in the art including, but not limited to,well-established spectrophotometric methods, such as ultraviolet (UV)and nuclear magnetic resonance (NMR) spectroscopy, and gelelectrophoresis. Shifts in UV spectra of nucleic acids are observed uponbinding of a MGB molecule, as are changes in NMR spectra, analyzedutilizing the Nuclear Overhauser (NOSEY) effect. Gel electrophoresisdetects binding of a MGB to double-stranded nucleic acid, because uponsuch binding the mobility of the double stranded nucleic acid changes.

As noted above, for the purposes of the invention, a molecule is a MGBif its association constant within the minor groove of a double strandednucleic acid is 10³ M⁻¹ or greater. However, certain MGBs bind to highaffinity sites with an association constant on the order of 10⁷ to 10⁹M⁻¹.

Thus, both structural and functional guidelines for the identificationof MGB moieties have been provided.

In addition to the molecular structure which causes minor groovebinding, the MGB moiety can also comprise additional functions, as longas those functions do not interfere with minor groove binding ability.

In accordance with the present invention, the MGB molecule isderivatized, i.e., formed into a radical, and linked to appropriatechains of atoms that attach the MGB to the ODN and/or to the LF. Theradical formed from the MGB molecule is hereinafter referred to as the“MGB moiety,” and the covalent linker (which can be a chain having abackbone of up to approximately 100 atoms) that attaches the MGB moietyto the oligonucleotide or to the latent fluorophore is called the“linking group.” Preferred MGB moieties are described in U.S. Pat. No.5,801,155.

In a preferred embodiment, the minor groove binder moiety is covalentlyattached to either the 3′- or 5′-end of the oligonucleotide, through aterminal base, sugar or phosphate moiety, or through a tail moietyattached to one of these moieties. In additional embodiments, the MGB isattached to a nucleotide in an internal position, particularly to thebase portion of the nucleotide.

Latent Fluorophores

The invention provides compositions and methods, involving the use oflatent fluorophores, for detection of nucleic acids byhybridization-triggered fluorescence. A latent fluorophore is a moleculein which a physical property of the fluorophore is altered by itsinteraction with duplex or triplex nucleic acids, resulting in a changein the fluorescence spectrum and/or an increase in the fluorescencequantum yield at a particular wavelength, and/or a change in some otherfluorescent property of the molecule. A change in fluorescence spectrumcan include a change in the absorption spectrum and/or a change in theemission spectrum.

The majority of interactions between multi-stranded nucleic acids andtheir ligands can be described in terms of two types of bindinginteractions: intercalation and groove binding. Groove binding includesboth major groove binding and minor groove binding. All of these bindinginteractions can be exploited in the design of latent fluorophores. Forexample, intercalation within a double-stranded DNA molecule can resultin a decrease in the rotational freedom of a ligand, and/or a change inthe dielectric environment that the ligand experiences. The inventionprovides examples of hybridization-triggered enhancement in quantumyield resulting from both intercalation and groove binding. Examples oflatent fluorophores and methods for determining whether a molecule hasthe properties of a latent fluorophore are also provided.

Certain cyanine dyes (see FIG. 1 for exemplary structure) are virtuallynon-fluorescent in the absence of nucleic acid. When free in solutionthese compounds transit from the excited singlet state (S₁) to theground state (S₀) in a radiationless process involving loss ofexcitation energy by rotation about the cyanine methine bond (FIG. 1A).Cyanine dyes interact with double-stranded nucleic acid byintercalation. Intercalation prevents free rotation about the cyaninemethine bond and causes the dye to lose excitation energy byfluorescence emission (FIG. 1B). Thus, without wishing to be bound bytheory, a potential mechanism for hybridization-triggered fluorescencerelates to restriction of rotation within a latent fluorophore followinginteraction with nucleic acid. Accordingly, molecules having these orsimilar properties are potential latent fluorophores.

In another aspect of the invention, hybridization-triggered increases influorescence quantum yield (or other changes in fluorescence properties)result from a change in the environment experienced by the latentfluorophore as a result of an interaction with double- ortriple-stranded nucleic acid. For example, a fluorescent reporter groupwill experience a more hydrophobic environment (i.e., a decrease indielectric constant) when intercalated or when positioned in the minoror major groove of a double-stranded nucleic acid. PRODAN(6-propionyl-2-dimethylaminonaphthalene) and2-(dimethylamino)naphthalene-6-sulfonamide are examples of fluorogenicreporter groups having structural features such that their quantum yieldand/or absorption maxima and/or emission maxima are sensitive to thistype of change in environment. Compounds such as these have a largedipole moment in the excited state, as a consequence of chargedelocalization between an electron-donating group and anelectron-accepting group. Exemplary electron-donating groups include,but are not limited to, N or O atoms having an electron pair availablefor extended charge localization, for example, RO— and (R₁)(R₂)N—,wherein R, R₁ and R₂ are independently H or alkyl, and wherein R₁ and R₂can also be part of a 5- or 6-membered ring system. Exemplaryelectron-accepting groups include, but are not limited to, —NO₂,—C(═O)—, —C(═S)—, —C(═O)—NH—, —CN, —N(═O), —S(═O)₂—, —S(═O)₂—NH—, and—C═C(CN)₂. The group (−)N═C(−)(−) can also serve as anelectron-accepting group, wherein N and C can both be part of a ringsystem or C alone can be part of a ring system. In general,electron-donating and -accepting groups and their properties arewell-known to those of skill in the art.

Additional environment-sensitive fluorogenic species, capable ofdelocalizing electron density via conjugated electron donor-electronacceptor groups, include derivatives of2-dimethylaminonaphthalene-6-sulfonamides and the isomeric species5-dimethylaminonaphthalene-1-sulfonamides,4-(N-methylamino)-7-nitro-2,1,3-benzoxadiazole,6-anilinonaphthalene-2-sulfonamides, derivatives of pyridyloxazoles,1-ailinonaphthalene-8-sulfonic acid, 2-anilinonaphthalene-6-sulfonicacid, 2-(p-toluidinyl)naphthalene-6-sulfonic acid,N-phenyl-1-naphthylamine, thiazole orange, oxazole yellow, thiazoleblue, thiazole green, 4-(dicyanovinyl)julolidine,4-dimethylamino-4′-nitrostilbene, Nile Blue and Nile Red. See, forexample, Haugland, supra.

Compounds such as the aforementioned and their derivatives, whosefluorescence properties (such as quantum yield, absorption maximumand/or emission maximum) are sensitive to the polarity of theirenvironment, can be coupled to a linking group for attachment to a MGB(see below) and used as latent fluorophores in the practice of theinvention. As one example of the use of this type of latent fluorophore,Table 2, infra, shows an increase in fluorescence quantum yield for anoligonucleotide-MGB-(2-dimethylaminonaphthalene-6-sulfonamide) conjugateupon hybridization to a complementary DNA strand (conjugate #3, see alsoFIG. 5).

A number of commercially-available compounds, which exhibitenvironment-sensitive fluorescence after conjugation, containing varioustypes of reactive groups, are also useful. These include6-acryloyl-2-dimethylaminonaphthalene (acrylodan) and4-fluoro-7-nitrobenzofurazan (NBD). In the synthesis of ODN-MGB-LFconjugates, their reactive group can be reacted with nucleophilicgroups, for conjugation to a MGB moiety, by methods known to those ofskill in the art. See, for example, Casas-Finet et al. (1992) Proc.Natl. Acad. Sci. USA 89:1050-1054.

Additional examples of latent fluorophores, which can be attached toODN-MGBs using methods known in the art (e.g., Haugland, supra) include:

(1) derivatives of the structures represented by Formula 1

wherein R₉ and R₁₀ are independently —H or —(CH₂)_(m)CH₃ where m=0 to 5,or R₂₅, or R₉ and R₁₀ together form a 5- or 6-membered ring systemcontaining one or more C, N, O and/or S atoms;

R₁₁ contains one or more of the electron-withdrawing groups —C(═O)—,—C(═O)—O—, —C(═O)—NH—, —C(═S)—NH—, —N═N—, —S(═O)—, —S(═O)₂—,—S(═O)₂—NH—;

R₂₅ is —H or a linking group comprising a reactive group that reactswith hydroxyl, amino or sulfhydryl nucleophiles, and has a backbonebetween 1 and about 50 atoms long, wherein R₂₅ can contain the atoms H,C, N, O P and/or S, and wherein R₂₅ can contain one or more of thegroups —S—, —NH—, —O—, —NH—C(═O)—, —NH—C(═O)—NH—, —NH—C(═S)—,—NH—C(═S)—NH—, —O—P(═O)₂—O—NH—, —O—P(═O)₂—O—; and

each of R₁₂ is independently R₂₅, —H, a halogen; NO₂; —COOH; —CONH₂,—CONHR₆; —CON(R₆)₂; —OR₆; —SO₃H; —SO₂NH₂; —SO₂NHR₆; —SO₂N(R₆)₂; —SR₆;—R₆; C(═O)—O—R₆; or —N(R₉)(R₁₀);

wherein R₆ is —(CH₂)_(m)CH₃ where m=0 to 5;

wherein R₉ and R₁₀ are defined as above.

(2) derivatives of the structures represented by Formula 2

wherein Z is —O— or —S—;

n is between 0 and 5;

Y is H, —(CH₂)_(n)CH₃ where m=0 to 4, or R₂₅, wherein R₂₅ is defined asin Formula 1; and

R₁₂ is defined as in Formula 1.

(3) thiazole-indoline derivatives as shown in Formula 3

wherein X is —O— or —S—;

n is between 0 and 5;

Y is defined as in Formula 2; and

R₁₂ is defined as in Formula 1.

(4) derivatives of 4-(N-methylamino)-7-nitro-2,1,3-benzoxazole asrepresented by Formula 4

wherein R₁₈ and R₁₉ are independently R₉, R₁₀, R₁₁R₂₅ or R₂₅, where R₉,R₁₀, R₁₁ and R₂₅ are defined as in Formula 1.

(5) derivatives of the structures represented by Formula 5

wherein R₁₂ is defined as in Formula 1; and

R₂₀ is —H, —(CH₂)_(m)CH₃ where m=0 to 5, or R₂₅, where R₂₅ is defined asin Formula 1.

(6) derivatives of the structures represented by Formula 6

wherein R₁₂ is defined as in formula 1 and R₂₀ is defined as in Formula5.

(7) derivatives of the structures represented by Formula 7

wherein R₂₀ is defined as in Formula 5.

(8) derivatives of the structures represented by Formula 8

wherein R₂₀ is defined as in Formula 5.

(9) derivatives of the structures represented by Formula 9

wherein R₂₅ is defined as in Formula 1.

(10) derivatives of the structures represented by Formula 10

wherein R₁₂ is defined as in Formula 1 and R₂₀ is defined as in Formula5.

(11) derivatives of the structures represented by Formula 11

wherein R₉, R₁₀ and R₁₂ are defined as in Formula 1; and

R₂₀ is defined as in Formula 5.

In one embodiment, a latent fluorophore is covalently linked to a MGBand/or an ODN via one or more linking groups. A linking group can beR₂₅, wherein R₂₅ comprises a backbone of from 1 to about 50 atoms,preferably 40 atoms, more preferably 30 atoms, more preferably 20 atoms,still more preferably 10 atoms and most preferably 5-6 atoms containingC, H, N, O, S and/or P atoms, and comprises one or more of the groups—S—, —NH—, —O—, —NH—C(═O)—, —NH—C(═O)—NH—, —NH—C(═S)—, —NH—C(═S)—NH—,—O—P(═O)₂—O—NH— and —O—P(═O)₂—O—. See infra for further discussion oflinking groups. In additional embodiments, linkage between a LF and aMGB and/or an ODN is via the groups R₁₁R₂₅, wherein R₁ includes anelectron-withdrawing group such as, for example, —C(═O)—, —C(═O)—O—,—C(═O)—NH—, —C(═S)—NH—, —N═N—, —S(═O)—, —S(═O)₂— and —S(═O)₂—NH—, andR₂₅ is defined as described supra. When the configuration of theconjugate is ODN-MGB-LF, the LF is linked to the MGB by a single linkinggroup; when the configuration of the conjugate is ODN-LF-MGB, twolinking groups are attached to the LF: one to the ODN and one to theMGB.

The invention has identified structural features in organic moleculesthat qualify them as potential latent fluorophores. The general featuresof candidate compounds are shown below:

A candidate latent fluorophore thus requires three different structuralfeatures, designated I, II and III above. I and III are respectivelyelectron donating and electron accepting groups connected to structuralfeature II, a resonance linker which, by allowing interaction betweengroups I and III, permits extended charge localization with large dipolemoments. Electron-donating and electron-accepting groups are well knownin the art. Exemplary electron-donating groups include N or O atoms withan electron pair available for extended localization, e.g. RO— or(R₁)(R₂)N—, wherein R, R₁ and R₂ are independently H or alkyl andwherein R₁ and R₂ can together form a 5- or 6-membered ring system.Exemplary electron-accepting groups include, but are not limited to—NO₂, —C(═O)—, —C(═S)—, —C(═O)—NH—, —CN, —N(═O), —S (═O)₂—, —S(═O)₂—NH—, —C═C(CN)₂ and (−)N═C(−)(−) wherein N and C can be part of aring system. Resonance linker groups include aromatic ring systemsand/or conjugated double and triple bond moieties. Structural features Iand III are separated by at least one conjugated double or triple bond.

In another embodiment, methods for identification ofenvironment-sensitive fluorophores are provided. A compound is tested bydetermining its fluorescent spectra in four solvents with differentpolarities. Solvents having the requisite properties will be apparent tothose of skill in the art. In one embodiment, the solvents are water,methanol, ethanol and ethyl acetate; having dielectric constants of78.54, 32.6, 24.3 and 6.02, respectively. As an example, thefluorescence intensities of a number of known LFs were evaluated inwater and in ethanol as shown in Table 1. Based on these results, acompound whose fluorescent signal in ethyl acetate, ethanol or methanolis about six-fold or greater that its fluorescent signal in water is acandidate latent fluorophore. It is likely that even smaller differencesin fluorescence between different solvents, i.e., on the order of two-or three-fold, is indicative of a candidate LF. Further evaluation of acandidate LF is accomplished by synthesis of its ODN-MGB conjugate andtesting for hybridization-triggered fluorescence. In addition, acompound that exhibits changes in fluorescence excitation and/oremission maxima in less polar solvents, instead of or in addition to anincrease in fluorescence quantum yield, is also a potential latentfluorophore.

TABLE 1 Fluorescence of known latent fluorophores in water and ethanol.Fluorescent Intensity (FL) Compound λ (nm) Water EthanolFL_(EtOH)/FL_(H2O)

405 69 412 6

480 8 181 23

525 4 226 57

538 2 674 337

445 17 531 31

Environment sensitivity of fluorescence was tested for two relatedcompounds, one of which (Compound A) contained structural features I, IIand III as described above, and one of which (Compound B, a reducedderivative of Compound A) did not. These compounds were synthesizedaccording to Boger et al. (1987) J. Org. Chem. 52:1521-1530. Aspredicted on the basis of its structural features, Compound A exhibiteda 31-fold difference in fluorescence emission between its water andethanol solutions. Reduced derivative B showed only a two-folddifference under similar conditions. In light of the results presentedin Table 1, the environment-sensitive characteristics of Compound Asuggest its use as a latent fluorophore.

Preferred embodiments of ODN-MGB-LF conjugates are those in which thelatent fluorophore is covalently attached to the MGB and/or the ODN in amanner that maintains or enhances its ability to undergohybridization-triggered fluorescence; for example, by allowingrotational freedom between the LF and the remainder of the conjugate.Methods for attachment of fluorophores to MGB and/or ODN moieties inthis manner, and the chemical principles involved, are known in the artand are described infra and, for example, in Haugland, supra.Furthermore, the optimal structural relationship between a LF and theother components of the conjugate is one that results, uponhybridization, in projection of the LF into a non-polar region or into aregion that restricts the rotational freedom of the LF, resulting inincreased fluorescence.

Linking Groups

The ODN, MGB and LF moieties are covalently joined to one another byvarious linking groups. In one configuration, conjugates of theinvention have the structure ODN-MGB-LF. For this configuration,preferably the linking groups are such that the linkage between the ODNand the MGB occurs through a chain of no more than about 100 atoms,preferably 80, more preferably 60, more preferably 40, more preferably20, still more preferably 10, and most preferably about 5-6 atoms, andthe linkage between the MGB and the LF occurs through a chain of no morethan about 50 atoms, preferably 40 atoms, more preferably 30 atoms, morepreferably 20 atoms, still more preferably 10 atoms and most preferablyabout 5-6 atoms. Another configuration of the conjugates of theinvention has the structure ODN-LF-MGB. In this configuration, thelinkage between the ODN and the LF occurs through a chain of no morethan about 50 atoms, preferably 40 atoms, more preferably 30 atoms, morepreferably 20 atoms, still more preferably 10 atoms and most preferablyabout 5-6 atoms and the linkage between the LF and the MGB occursthrough a chain of no more than about 50 atoms preferably 40 atoms, morepreferably 30 atoms, more preferably 20 atoms, still more preferably 10atoms and most preferably about 5-6 atoms.

Generally speaking, the linking group is derived from a bifunctionalmolecule such that one functionality (e.g., an amine) is attached, forexample, to a 5′ phosphate end of an ODN, and the other functionality(e.g., a carbonyl group) is coupled, for example, to an amino group of aminor groove binder moiety. Alternatively, a linking group can bederived from an amino alcohol so that the alcohol function is linked,for example, to a 3′-phosphate end of an ODN and the amino function islinked, for example, to a carbonyl group of a MGB moiety. Additionallinking groups include amino alcohols (attached, for example, to the3′-phosphate of an ODN via an ester linkage) linked to anaminocarboxylic acid which in turn is linked in a peptide bond to acarbonyl group of a MGB. See U.S. Pat. No. 5,801,155 for furtherdisclosure related to linking groups. Thus, preferred embodiments of thelinking group have backbones containing the atoms C, N, O, P and/or Sand can contain one or more of the groups —NH—, —O—, —C(═O)—, —O—C(═O)—,—NH—C(═O)—, —C(═O)—NH—, —NH—C(═O)—NH—, —NH—C(═S)—, —NH—C(═S)—NH—, —N═N—,—O—P(═O)₂—NH—, —O—P(═O)₂—O—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH—, —S—, and—S—S—. Preferably the MGB moiety is separated by not more thanapproximately 100 atoms from the ODN and not more than approximately 50atoms from the LF. Accordingly, more preferred embodiments of linkinggroups include, for example, —(CH₂)₃C(═O)NH(CH₂)₆C(═O)— and —O(CH₂)₆NH—.

As mentioned supra, the presence of a latent fluorophore renders acomposition readily detectable by an increase or decrease in adiscernible physical or chemical characteristic upon hybridization to atarget sequence. In one embodiment, a latent fluorophore is covalentlyattached to a minor groove binder moiety by a linking group. The2-dimethylaminonaphthalene-6-sulfonyl function is an example of apreferred embodiment of a latent fluorophore, which can be attached to acarbonyl function of the minor groove binder through a —HN(CH₂)_(m)NH—bridge, where m is such that the length of the linker between the MGBand the LF is no more than about 50 atoms. The latent fluorophore can becoupled to one end of this bridge by chemistries known in the art, forexample through the use of coupling groups such as —C(═O), —O—C(═O)—,—NH—C(═O)—, —NH—C(═S)— and —CH₂—.

Alternatively, a reactive group can be attached directly to a LF tofacilitate its coupling to a linking group of a MGB or ODN. Suchreactive groups include, but are not limited to, moieties such ascarbonates, isocyanates, isothiocyanates, mono- or di-substitutedpyridines, maleimides, aziridines, acid halides, sulfonyl halides,monochlorotriazines, dichlorotriazines, hydroxysulfosuccinimide esters,hydroxysuccinimide esters, azidonitrophenyls, azides, aldehydes,ketones, glyoxals and 3-(2-pyridyl dithio)-propionamide.

Hybridization-triggered Fluorescent Probes for Detection ofDouble-stranded Nucleic Acids

ODN-MGB-LF conjugates can be used for detection of both single-strandedand double-stranded nucleic acid targets. For detection ofdouble-stranded nucleic acids, the oligonucleotide component of theconjugate is a triplex-forming oligonucleotide (TFO), and binds in themajor groove of the double stranded target via Hoogsteen, reverseHoogsteen or equivalent base pairing, as is known in the art. The MGBcomponent of the conjugate binds to the minor groove of thedouble-stranded target. Synthesis of conjugates capable of simultaneousbinding of the TFO in the major groove and the MGB in the minor grooveis accomplished by attaching the MGB to the TFO via a long flexiblelinker, having a length up to about 100 atoms, such that the flexiblelinker is able to wrap around one of the strands of the duplex target.TFO-MGB conjugates of this kind have been described. Lukhtanov et al.(1997a) J. Am. Chem. Soc. 119:6214-6225; and Lukhtanov et al. (1997b)Nucleic Acids Res. 25:5077-5084. In a TFO-MGB-LF conjugate designed fordetection of a double-stranded target, the latent fluorophore will beanchored in the minor groove and will undergo either an increase influorescence intensity at a given wavelength or some other discernablechange in fluorescent properties as described supra.

The MGB-LF portion of the conjugates can also gain access to the minorgroove of target double-stranded DNA by threading through the base-pairstack, from the major to the minor groove. The threading phenomenon hasbeen previously described in the literature, mostly associated withthreading intercalators which are intercalating moieties bearing bulkyside chains that can pass through the base pair stacks of duplex nucleicacids. The Pluromycins, which are known to thread the DNA structure,placing carbohydrate residues into both grooves, provide an example.Hansen et al. (1996) Acc. Chem. Res. 29:249-258.

Synthesis of MGB-ODN-LF Conjugates

Preferred embodiments of minor groove binder moieties are oligopeptidesderived from 1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid(CDPI) and from N-methylpyrrole-4-carbox-2-amide (MPC). These have beendescribed in detail in U.S. Pat. No. 5,801,155, wherein a process wasdisclosed for preparing the tripeptide CDPI₃, which thereafter can becoupled, in accordance with the present invention, and with or withoutminor modification, to an ODN to form a portion of a preferredODN-MGB-LF conjugate.

In Reaction Scheme 1, a general method for coupling a 3′-amino-tailed or5′-amino-tailed ODN with a tetrafluorophenyl (TFP) ester of an exemplaryminor groove binding oligopeptide is illustrated. The scheme shows theuse of a TFP-activated exemplary minor groove binding compound obtainedin

accordance with U.S. Pat. No. 5,801,155; however, this general method issuitable for the coupling of any TFP-activated minor groove bindingcompound to an ODN. Reference numerals 1a and 1b in Reaction Scheme 1refer to exemplary compounds obtained in accordance with methodsdescribed in U.S. Pat. No. 5,801,155, the disclosure of which isexpressly incorporated herein by reference.

A 5′- or 3′-amino-tailed ODN can be synthesized by conventional methods;for example an aminohexyl residue can be attached to either end of anODN by using commercially available MMT-aminohexyl phosphoramidite (5′tail) or N-Fmoc-aminohexyl-CPG (3′ tail). Alternatively, an amino-tailedODN can be synthesized in accordance with the methods described in U.S.Pat. No. 5,419,966, the disclosure of which is expressly incorporatedherein by reference. In accordance with the present scheme, theamino-tailed ODN is converted into a cetyltrimethylammonium salt torender it soluble in organic solvents, and the tetrafluorophenylester-activated MGB molecule is condensed therewith, preferably usingDMSO as a solvent.

Reaction Scheme 2 discloses another method for coupling an active esterof a minor groove binder molecule to a 5′- or 3′-amino tailed ODN (2).

The TFP ester of the tripeptide (n=3) derived fromcarbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid(TFP-CDPI₃) is shown as an exemplary MGB; however, it will be clear toone of skill in the art that the generic principles disclosed inconnection with this reaction scheme can be used with other minor groovebinder molecules as well. In this method, the ODN comprises a tailmoiety (wherein m=1 to 99) comprising a free terminal amino group, andremains attached to a CPG support during the addition of the MGB. Suchan ODN is obtained, for example, by stepwise synthesis on a CPG support,using a MMT-aminohexyl phosphoramidite in the terminal addition step.This generates a CPG-bound ODN having a 5′ tail comprising an aminogroup protected with a monomethoxytrityl (MMT) group. After synthesis ofthe ODN is complete, the MMT group is removed from the amino group usingconditions under which the ODN remains attached to the CPG support, forexample, by treatment with 3% trichloroacetic acid in CH₂Cl₂. Inaccordance with Reaction Scheme 2, the free amino group of thisCPG-bound, amino-tailed-ODN is condensed with an active ester (e.g.,TFP-CDPI₃, 1b) or with a similarly activated form of a minor groovebinder. The ODN-MGB conjugate is thereafter removed from the CPG supportby conventional methods, preferably by treatment with ammonia.Alternatively, a CPG-bound, 3′-amino-tailed ODN is obtained inaccordance with the disclosure of U.S. Pat. No. 5,419,966, andreferences cited therein.

Another exemplary protecting group is the 9-fluorenylmethoxycarbonyl(Fmoc) group, which is removed by base treatment, as is known to thoseof skill in the art. Additional protecting groups, such as carbamateprotecting groups, amide protecting groups and a series of specialprotecting groups are described in Green, T. W. & Wuts, P. G. M. inProtective Groups in Organic Synthesis, 2^(nd) Edition, John Wiley andSons, Inc, NY., pp. 441-452. 1991.

Synthesis of 1-(3-hydroxypropyl)-thiazole orange (Compound 4 whereinq=3) was carried out in two steps, using methodology similar to thatused for the synthesis of 1-(3-iodopropyl)-thiazole orange (ReactionScheme 3). Benson et al. (1993) Nucleic Acids Res 21:5727-5735; Brookeret al. (1941) J. Am. Chem. Soc. 63:3192-3202; and Brooker et al. (1942)J. Am. Chem. Soc. 64:199-210. Conversion to the activated 4-nitrophenylcarbonate derivative (5) was accomplished by the reaction of 4 with4-nitrophenyl chloroformate. Alternatively, a reactive group can beintroduced at the 3-position of 2-(methylthio)-1,3-benzothiazole usingreactions described by Brooker et al. (1941, 1942), supra. In addition,substituents such as —H, -halogen, —NO₂, —SO₃H, —COOH, —CONHR₆,—CON(R₆)₂, —OR₆, —SO₂NHR₆, —SO₂N(R₆)₂ and —SR₆; wherein R₆=—(CH₂)_(m)CH₃and m=0 to 5; can be introduced on either ring of compound 3 as well asthe phenyl ring of 2-(methylthio)-1,3-benzothiazole.

Another preferred method for preparing a ODN-MGB-LF conjugate is shownin Reaction Schemes 4 and 5. Reaction Scheme 4 shows the synthesis of aMGB with reactive groups at both ends (12) for use in Reaction Scheme 5.The amino group of 6-aminohexanoic acid (n=5) was blocked with a MMTrgroup to form intermediate 6, whose carboxylic acid group was thenactivated with tetrafluorophenyltrifluoroacetate to yield intermediate7. Reaction of 7 with methyl pyrrolo[4,4-e]indoline-7-carboxylateyielded the methyl ester 8 which was converted to the acid 9. Reactionof 9 with3-(pyrrolo[4,5-e]indolin-7-ylcarbonyl)pyrrolo[4,5-e]indoline-7-carboxylate,followed by consecutive LiOH and TFP-TFA treatment yielded the CDPI₃conjugate (12) containing a terminal MMTr-protected amino group and aTFP-protected ester at the other terminus. Conjugation of novel reagent12 at one of its ends to an ODN and the other of its ends to a LF ispossible by virtue of its terminal reactive groups.

For example, in Reaction Scheme 5, conjugate 12 is reacted with an ODNcontaining a 5′-aminoalkyl group to yield intermediate 13. Removal ofthe MMTr group with 80% acetic acid, and subsequent reaction with theactivated carbonate (5) from Reaction Scheme 3, yielded the ODN-CDPI₃-TO(thiazole orange) conjugate 14. It is clear that similar reactions canbe used to introduce different linkers between the MGB and the ODN andLF, respectively, to generate conjugates with the general formulaindicated by Formula 12, where each of n and q is at least one, the sumof n and q is no greater than 46, and m=1-99.

Furthermore, it is clear to those of skill in the art that a number ofdifferent MGBs and LFs, as disclosed herein, can be used in thereactions described above, to generate a wide variety of ODN-MGB-LFconjugates of this particular configuration.

Reaction scheme 6 discloses another preferred method for preparing a3′-ODN-MGB-LF conjugate. Intermediate 15 is synthesized by amodification of the methods disclosed in U.S. Pat. No. 5,801,155, asshown in Reaction Scheme 7. After deprotection with TCA/CH₂Cl₂, the CPGderivative was used for standard oligonucleotide synthesis to obtain therequired oligonucleotide sequence. Cleavage of the ODN from the CPG withammonia yielded intermediate 16, which was coupled to an amine-reactivelatent fluorophore to give the desired ODN-MGB-LF conjugate 17.

Intermediate 15 was prepared as shown in Reaction Scheme 7, startingwith the reaction of p-nitrophenyl chloroformate with2,2′-sulfonyldiethanol to yield 18. This compound was successivelyreacted with (3-aminopropyl)[(4-methoxyphenyl)diphenylmethyl]amine andactivated with p-nitrophenyl chloroformate to yield 20. After thereaction of 20 with long chain amino CPG, deprotection with TCA/CH₂Cl₂and reaction with activated ester 24, intermediate 22 was obtained. TFAdeprotection of 22 followed by reaction with 25 gave intermediate 23which was deprotected with TFA and reacted with p-nitrophenyl-4-O-DMTbutyrate to provide the desired intermediate 15.

More generally, intermediates equivalent to compound 15 can be used forsynthesis of ODN-MGB-LF conjugates. These intermediates contain acleavable linker K between the CPG moiety and the MGB moiety, as shownin Formula 13 below:

A variety of cleavable linkers useful for interposition between a CPGand a MGB, as shown in formula 13 by K, are known in the art. Theseinclude, but are not limited to, phosphodiester groups modified with alinker bearing an amino, thiol or hydroxyl group, andhydroquinone-O,O′-diacetic acid linkers. Lyttle et al. (1997) Bioconjug.Chem. 8:193-198; and Pon et al. (1997) tetrahedron 39:3327-3330. CPGsupports with attached cleavable linkers are also available and include,for example, universal solid suppports and long-chainalkylamidopropanoic acid CPG. Scott et al. (1994) “Innovations andPerspectives in solid Phase Synthesis” 3^(rd) International Symposium,ed. R.Epton, Mayflower Worldwide, pp. 115-124; and Damha et al. (1990)Nucleic Acids Res. 18:3813-3812.

In another embodiment, the LF can be incorporated on the linker betweenthe ODN and MGB, rather than as shown in compound 17, Reaction Scheme 5,where the ODN and LF are on opposite ends of the MGB. To achieve this,Reaction Scheme 5 is modified, such that the ODN contains anappropriately-blocked hydroxyalkyl amine group at its 5′ end. The aminogroup, after deprotection, is used to attach the MGB; and the hydroxylgroup, after deprotection and activation, is used to attach the LF. Forexample, the CPG-(CDPI)₃-DMTr intermediate described by Lukhtanov et al.(1996) Bioconj. Chem. 7:564-567 is reacted with the phosphoramidite of2-(4-Fmoc-aminobutyl)-1-(DMTrO)-propane-3-ol (Clontech, Palo Alto,Calif.), followed by standard oligonucleotide synthesis. After synthesisof the desired oligonucleotide is complete, cleavage from the CPG,followed by removal of the Fmoc blocking group, allows attachment of aLF to the amino group of the linker using reagent 5.

In another embodiment, the LF is attached at a site internal to the MGB,as follows. Reaction scheme 4 can be modified such that7-(methoxycarbonyl)-4-[(phenylmethoxy)carbonylamino]pyrrolo[3,2-e]indoline-2-carboxylicacid (Boger et al. (1992) J. Org. Chem. 57:1277-1284) is reacted withmethyl3-(pyrrolo[4,5-e]indolin-7-ylcarbonyl)pyrrolo[4,5-e]indoline-7-carboxylate(Boger et al., supra) in the presence of a coupling reagent to form theequivalent of 10, which after H₂/Pd/C treatment yields methyl2-[2-{[3-[{5-amino-3-[(tert-butyl)oxycarbonyl]pyrrolo[4,5-e]indolin-7-yl}carbonyl]pyrrolo[4,5-e]indolin-7-yl]carbonyl}-3-pyrrolino[3,4-e]indolin-8-yl]acetate.This compound contains a free primary amino group which can be used forattachment of the LF, a t-Boc-protected nitrogen and a methylester-protected carboxylic acid. Either of the protected groups can beused for attachment of the oligonucleotide.

Characteristics of Hybridization-Triggered Fluorescence with ODN-MGB-LFConjugates

Free cyanine dyes, such as TO, bind to double- and triple-strandednucleic acid in a non-sequence-specific fashion or, at best, with onlybroad sequence preferences. By contrast, cyanine dyes and other latentfluorophores, when present in an ODN-MGB-LF conjugate, interact withnucleic acid based upon hybridization of the ODN portion of theconjugate with its complementary target. Thus, unlike free(unconjugated) dyes, ODN-MGB-LF conjugates bind with high specificity tosequences complementary to their ODN portion, and are capable ofdiscriminating between closely-related DNA sequences with similar hybridmelting temperatures.

For example, an exemplary latent fluorophore is the cyanine dye thiazoleorange (TO), which becomes highly fluorescent upon intercalation intodouble-stranded DNA. However, free TO binds in a sequence-independentfashion to double-stranded DNA, and thus cannot be used as asequence-specific diagnostic probe. However, as part of an ODN-MGB-LFconjugate, the fluorescent potential of TO is coupled with the sequencespecificity imparted by the oligonucleotide, to obtain sequence-specificfluorescent detection of a complementary target sequence.

Hybridization-triggered fluorescence, using the methods and compositionsof the invention, can be obtained for target sequences that are eitherAT- or GC-rich. FIGS. 2A and 2B provide examples in which a cyanine dye(TO) is used as a latent fluorophore in an ODN-MGB-LF conjugate todetect an AT-rich target sequence (FIG. 2A) and a GC-rich targetsequence (FIG. 2B). FIGS. 2A and 2B show that the ODN-MGB-TO conjugateexhibits an increase in fluorescence emission intensity only afterspecific hybridization with a complementary target sequence.

In the example shown in FIGS. 2A and 2B, restricted rotation about thecyanine-methine bond of the TO molecule is believed to be responsiblefor the increase in fluorescence quantum yield. Without wishing to bebound by any particular theory, it is thought that restriction ofrotation is a result of intercalation of the TO molecule intodouble-stranded DNA. For latent fluorophores other than TO, binding toDNA can result in restrictions of rotational freedom by othermechanisms, such as major groove or minor groove binding, or bymechanisms resulting from the conjugation of the latent fluorophore tothe MGB-ODN and base-pairing of the ODN with its complementary targetsequence.

Attachment of a latent fluorophore to a MGB moiety facilitates theobserved increase in fluorescent output by a latent fluorophorefollowing hybridization of an ODN-MGB-LF conjugate to a complementarytarget sequence. This is demonstrated in FIGS. 3A and 3B, which showchanges in fluorescent output for ODN-TO conjugates containing (FIG. 3A)or lacking (FIG. 3B) a MGB as part of the conjugate. Without wishing tobe bound by any particular theory, it is thought that the anchoring ofthe MGB moiety of the conjugate in the minor groove facilitatesintercalation by the LF (in this case, the TO moiety) and subsequentfluorescence. Additional mechanisms, such as synergistic interactionsbetween the MGB and the LF, are also possible.

Additional examples of hybridization-triggered fluorescence arepresented in Table 2, in which different LFs and different ODNs wereevaluated. Hybridizations were conducted with 1×10⁻⁷ M conjugate and a2-fold molar excess of complementary target sequence in a pH 7.4phosphate buffer for 5 min at 25° C. (See Example 1 for buffercomposition.) Increase in fluorescence yield (“Fluorescence Increase”column of Table) is presented as the ratio of fluorescence emitted bythe hybrid between the ODN-MGB-LF conjugate and its target sequence tothe fluorescence emitted by unhybridized (i.e., single-stranded)ODN-MGB-LF.

TABLE 2 Hybridization-triggered fluorescence with different ODN-MGB-LFconjugates Schematic Representation of Fluorophore-MGB-ODN Conjugates

Conjugate R1 R2 Fluorescence Increase 1

TTTTTTTTTTTTTTTT(SEQ ID NO: 21) 8.3 2

GAAGTTGCTT(SEQ ID NO: 6) 3.1 3

GAATTTTGCTT(SEQ ID NO: 7) 4.2 4

TTTTTTTTTTT(SEQ ID NO: 22) 8.7 5

TTTTTTTTTTTTTTT(SEQ ID NO: 23) 23

An example of hybridization-triggered fluorescence in a DNA-RNA hybrid,using a ODN-MGB-LF conjugate, is provided in FIG. 4. This figure showsthat when a poly(dT)₁₅-MGB-dansyl conjugate (SEQ ID NO:24) is hybridizedto a poly(A) target, an approximately 8-fold increase in fluorescence,compared to unhybridized conjugate, is observed. Hybridizationconditions are given in the legend to FIG. 4. This result demonstratesthat hybridization-triggered fluorescence can be observed in hybridsbetween heterologous polynucleotides such as DNA and RNA, and is thus ageneral phenomenon.

In general, the T_(m) of a hybrid between an ODN-MGB-LF and its targetsequence is higher than the T_(m) of a hybrid between an unconjugatedODN and the same target sequence, due to the presence of the MGB. See,for example, U.S. Pat. No. 5,801,155. Consequently, at stringencies atwhich an unconjugated ODN is not able to form hybrids with sequencesrelated to its complementary target sequence (i.e., mismatches), anODN-MGB-LF may be capable of forming hybrids with such relatedsequences. Accordingly, ODN-MGB-LF conjugates can be used, not only fordetection of a perfectly complementary target sequence, but also fordetection of sequences related to a target sequence that iscomplementary to the ODN portion of the ODN-MGB-LF conjugate as, forexample, in the identification of gene families.

ODN-MGB-LF compositions are also useful in methods that involve mismatchdiscrimination. In this respect, they are similar topreviously-described ODN-MGB conjugates, which form highly stableduplexes with perfectly complementary sequences, but more unstableduplexes with target sequences containing a single-nucleotide mismatchwith respect to the ODN portion of the conjugate. This property ofODN-MGB conjugates is observed for ODN sequences at least as short as 8nucleotides. See International Patent Application No. PCT/US99/07487.However, unlike ODN-MGB conjugates, hybrids comprising ODN-MGB-LFconjugates are inherently detectable by virtue of theirhybridization-triggered fluorescence.

Mismatch detection by an ODN-MGB-LF conjugate is exemplified in FIG. 5,wherein it is shown that an ODN-MGB-LF (conjugate 3 of Table 2) does notexhibit substantial fluorescence when it is incubated underhybridization conditions with a sequence having a single-nucleotidemismatch with the ODN portion of the conjugate. Incubation of the sameODN-MGB-LF with a perfectly complementary target sequence under the sameconditions, however, as shown in FIG. 5, results in an increase influorescence. Hybridization conditions are given in the legend to FIG.5.

In another experiment, the melting temperatures (T_(m)s) of hybridsbetween ODN-MGB-LF conjugates, and either perfectly-matched orsingle-nucleotide mismatched DNA target sequences, were determined. Thiswas accomplished by forming hybrids, gradually heating the hybrids, andplotting −dF/dt (change in fluorescence with respect to time) vs.temperature. The T_(m) (also known as T_(max)) is the temperature atwhich maximum −dF/dt is observed. Conjugates having an ODN portion ofdifferent lengths were tested and the results are provided in Example 8infra. ODN-MGB-TO conjugates, having oligonucleotide portions between 10and 18 nucleotides in length, provided excellent discrimination betweenperfectly matched and mismatched target sequences, with ΔT_(m)s of 10°C. or greater, where ΔT_(m) is the difference in melting temperaturebetween a perfectly-matched hybrid and a hybrid containing a mismatch.See Example 8.

Exemplary Applications for ODN-MGB-LF Conjugates

Methods and compositions of the invention are useful in the detection ofspecific nucleic acid sequences by hybridization. For the purposes ofthe invention, the term “hybridization” refers to the interaction of twoor more nucleic acids to form a stable multi-stranded structure. For twoor more nucleic acids to interact by “specific hybridization,” themulti-stranded structure formed therefrom can be a duplex, triplex, orany other higher order structure wherein the interaction is mediated, atleast in part, by specific base-pairing. Base-pairing includes so-calledWatson-Crick pairing, involved in duplex formation, as well as Hoogsteenand reverse Hoogsteen pairing, which are involved in triplex formation.Nucleic acids, either target nucleic acids or the oligonucleotideportion of a ODN-MGB-LF, can be DNA, RNA, modified DNA, modified RNA, orany modified nucleic acid or nucleic acid analogue known to one of skillin the art. Nucleic acid analogues include, but are not limited to,peptide or polyamide nucleic acids (Nielsen et al. (1991) Science254:1497-1500), bicyclo nucleic acids (Bolli et al (1996) Nucleic AcidsRes. 24:4660-4667) 1-α-arabinofuranosyl-containing oligonucleotides(U.S. Pat. No. 5,177,196) and oligonucleotide analogues with sulfamatelinkages (U.S. Pat. No. 5,470,967). Nucleic acids can also be chimericmolecules containing different types of nucleotides and/or nucleotideanalogues within the same molecule such as, for example, PNA/DNAchimeras. See, for example, Nielsen, supra and Koch, supra.

ODN-MGB-LF conjugates can be used as probes, in which theirhybridization to a target sequence is detected, or as primers, in whichtheir hybridization to a target sequence is followed by polynucleotidesynthesis initiated from the 3′ terminus of the oligonucleotide portionof the conjugate, and the synthesized product (i.e., the extensionproduct) is detected.

A target sequence refers to a nucleotide sequence in a nucleic acidwhich comprises a site of specific hybridization for a probe or aprimer. Target sequences can be found in any nucleic acid including, butnot limited to, genomic DNA, cDNA and RNA, and can comprise a wild-typegene sequence, a mutant gene sequence, a non-coding sequence, aregulatory sequence, etc. A target sequence will generally be less thanabout 100 nucleotides, preferably less than about 50 nucleotides, andmore preferably, less than about 25 nucleotides in length.

Hybridization of a probe and/or a primer to a target sequence to form aduplex proceeds according to well-known and art-recognized base-pairingproperties, such that adenine base-pairs with thymine or uracil, andguanine base-pairs with cytosine. The property of a nucleotide thatallows it to base-pair with a second nucleotide is calledcomplementarity. Thus, adenine is complementary to both thymine anduracil, and vice versa; similarly, guanine is complementary to cytosineand vice versa. An oligonucleotide which is complementary along itsentire length with a target sequence is said to be perfectlycomplementary, perfectly matched, or fully complementary to the targetsequence, and vice versa. An oligonucleotide and its target sequence canhave related sequences, wherein the majority of bases in the twosequences are complementary, but one or more bases are deleted,inserted, transposed or noncomplementary (i.e., mismatched). In such acase, the sequences can be said to be substantially complementary to oneanother, if their degree of complementarity is sufficient to allowdetectable hybrid formation. The ability to detect a hybrid will dependupon the stringency of hybridization, as is known to those of skill inthe art. See infra. If the sequences of an oligonucleotide and a targetsequence are such that they are complementary at all nucleotidepositions except one, the oligonucleotide and the target sequence have asingle nucleotide mismatch with respect to each other.

Conditions for hybridization are well-known to those of skill in the artand can be varied within relatively wide limits. Hybridizationstringency refers to the degree to which hybridization conditionsdisfavor the formation of hybrids containing mismatched nucleotides,thereby promoting the formation of perfectly matched hybrids or hybridscontaining fewer mismatches; with higher stringency correlated with alower tolerance for mismatched hybrids. Factors that affect thestringency of hybridization include, but are not limited to,temperature, pH, ionic strength, and concentration of organic solventssuch as formamide and dimethylsulfoxide. As is well known to those ofskill in the art, hybridization stringency is increased by highertemperatures and/or lower ionic strengths. See, for example, Ausubel etal., supra; Sambrook et al., supra; M. A. Innis et al. (eds.) PCRProtocols, Academic Press, San Diego, 1990; B. D. Hames et al. (eds.)Nucleic Acid Hybridisation: A Practical Approach, IRL Press, Oxford,1985; and van Ness et al., (1991) Nucleic Acids Res. 19:5143-5151. Thedegree of stringency can be adjusted not only during a hybridizationreaction, but also in post-hybridization washes, as is known to those ofskill in the art.

Thus, in the formation of hybrids between an ODN-MGB-LF and its targetsequence, the ODN-MGB-LF can be incubated in solution, together with apolynucleotide containing the target sequence, under conditions oftemperature, ionic strength, pH, etc, that favor specific hybridization(i.e., duplex or triplex formation mediated by base-pairing).Alternatively, the ODN-MGB-LF can be immobilized on a solid support,which is contacted with a solution potentially containing apolynucleotide comprising a target sequence. In yet another embodiment apopulation of polynucleotides, one or more of which potentiallycomprises a target sequence, is immobilized on a solid support, which iscontacted with a solution containing one or more ODN-MGB-LF conjugates.A polynucleotide is a polymer of nucleotides and is not limited withrespect to length. Polynucleotides can comprise DNA, RNA, and DNA and/orRNA analogues. A polynucleotide can also comprise multiple types ofnucleotides or nucleotide analogues, i.e., DNA/RNA or DNA/PNA chimeras.

Hybridization conditions are chosen, in some circumstances, to favorhybridization between two nucleic acids having perfectly-matchedsequences, as compared to a pair of nucleic acids having one or moremismatches in the hybridizing sequence (i.e., high stringencyconditions). In other circumstances, hybridization conditions of reducedstringency can be chosen to allow hybridization between mismatchedsequences.

The degree of hybridization of an oligonucleotide or oligonucleotideconjugate to a target sequence, also known as hybridization strength, isdetermined by methods that are well-known in the art. A preferred methodis to determine the melting temperature (T_(m)) of the hybrid duplex.This can be accomplished, for example, by subjecting a duplex togradually increasing temperature and monitoring the denaturation of theduplex, for example, by absorbance of ultraviolet light, since UVabsorption increases with the unstacking of base pairs that accompaniesdenaturation. T_(m) can be defined as the temperature midpoint of thetransition in ultraviolet absorbance that accompanies denaturation.Another quantitative indicator of hybridization strength is T_(max),which is the temperature at which the maximum rate of unpairing of baseswith respect to time is observed, as a hybrid is subjected tosuccessively increasing temperature. Unpairing of bases can be measured,for example, by changes in UV absorbance or by changes in fluorescenceof a hybrid containing an ODN-MGB-LF. A higher T_(max) correlates withincreased hybridization strength. Further description of T_(max)determination is presented in Example 8, infra.

One method for distinguishing between two duplexes, if their T_(m)s areknown, is to conduct hybridization at a temperature that is below theT_(m) of the desired duplex and above the T_(m) of an undesired duplex.In this case, determination of the degree of hybridization isaccomplished simply by testing for the presence of hybridized probe.

Thus, in one embodiment, MGB-ODN-LF conjugates are used as probes orprimers for detection of specific nucleic acid sequences. Detection isaccomplished according to techniques known to those of skill in the artincluding, but not limited to, solution hybridization, blothybridization, in situ hybridization, nuclease protection, cDNAsynthesis, priming, and amplification. Amplification technology includesboth target amplification methods and signal amplification techniques.

Target amplification methods include, for example, polymerase chainreactions (PCR), NASBA, SSSR, rolling circle amplification (Lizardi etal. (1998) Nature Genet. 19:225-232), cleavase-based amplification(Sander et al. (1999) Electrophoresis 20:1131-1140) and relatedamplification technologies. In the various target amplification methods,ODN-MGB-LF conjugates can be used as either primers for the synthesis ofamplification products or as probes to detect the amplificationproducts.

Signal amplification techniques involve hybridization of a probe, havingtwo portions, to a target sequence. A first portion of the probe iscomplementary to a target sequence. A second portion of the probe has aplurality of sequence units, each of which is complementary to a labeledoligonucleotide; alternatively, the second portion is complementary toanother probe having a plurality of sequence units, each of which iscomplementary to a labeled oligonucleotide. See, for example, U.S. Pat.Nos. 5,124,246; 5,594,118 and 5,902,724. The compositions and methods ofthe invention, when used in conjunction with signal amplificationmethods, for example as labeled oligonucleotides, provide even greatersensitivity by virtue of their capacity for hybridization triggeredfluorescence.

Additional applications include gene expression analysis,single-nucleotide polymorphism analysis and sequence-basedidentification of organisms, including infectious organisms, usingRT-PCR, arrays, and array-PCR. Additional detection systems aredisclosed in International Patent Application Nos. PCT/US99/07487 andPCT/US99/07492, the disclosures of which are incorporated herein byreference.

Hybridization-triggered fluorescence, according to the invention, can beused in any system in which detection of a hybrid duplex or triplex isof interest, by using the appropriate ODN-MGB-LF conjugate as a primeror a probe. Non-limiting examples include:

1) Quantitation of a particular nucleic acid sequence in the presence ofother similar nucleic acid sequences,

2) Qualitative discrimination between two sequences having a singlenucleotide difference, and

3) Detection of a very small amount of a specific DNA sequence.

An additional application of ODN-MGB-LF conjugates is in real-timedetection of PCR products. Wittwer et al. (1997) Biotechniques22:176-81. Under appropriate conditions, an ODN-MGB-LF conjugate used asa PCR primer provides single-nucleotide mismatch discrimination in realtime. See FIG. 6 and Example 9, infra.

A particular advantage of the hybridization-triggered fluorescent probesis in the area of multiplex detection (i.e., detection and quantitationof more than one PCR product in the same reaction vessel). For example,for two distinct target sequences, one complementary to ODN-A and theother complementary to ODN-B, conjugation of, for example, thiazoleorange to ODN-A and thiazole blue to ODN-B allows simultaneous detectionand quantitation of both target sequences. Additional latentfluorophores, having distinct emission maxima, can be conjugated toadditional oligonucleotides, to enable multiplex detection of additionaldistinct target sequences. The potential for multiplex detection usingthe methods and compositions of the invention is limited only by theresolving power of the fluorescent detection system.

The methods and compositions of the invention are also useful inprocedures that utilize arrays of oligonucleotides, such as sequencingby hybridization and array-based analysis of gene expression. In theseprocedures, an ordered array of oligonucleotides of different sequencesis used as a platform for hybridization to one or more testpolynucleotides, nucleic acids or nucleic acid populations. Generally,an array comprises a set of distinct addresses, each of which containsan oligonucleotide of distinct sequence. Determination of theoligonucleotides that are hybridized and alignment of their sequences,if known, allows reconstruction of the sequence of the testpolynucleotide. See, for example, U.S. Pat. Nos. 5,492,806; 5,525,464;5,556,752; and PCT Publications WO 92/10588 and WO 96/17957. Materialsfor construction of arrays include, but are not limited to,nitrocellulose, glass, silicon wafers, optical fibers and othermaterials suitable for construction of arrays such as are known to thoseof skill in the art.

In a preferred array method, an ODN-MGB-LF conjugate is immobilized on asolid surface, where it serves as a capture probe and/or an extensionprimer. Hybridization and/or extension results in fluorescence. Variousmethods for immobilization of ODN conjugates to solid surfaces are knownin the art. See, for example, Ramsay (1998) Nature Biotechnol 16:40-44;U.S. Pat. No. 5,412,087; U.S. Pat. No. 5,424,186; WO 95/11748 and EP373,203.

ODN-MGB-LF conjugates are particularly advantageous for use asimmobilized probes in various types of array-based technology, becauseassays can be conducted without the necessity for labeling targetnucleic acids. Hybridization of a target nucleic acid to an immobilizedODN-MGB-LF on an array results in the immediate generation of afluorescent signal at the site of the hybridized probe, without the needfor any type of post-hybridization labeling or detection steps.

The following examples are provided to illustrate, but not to limit, theinvention.

EXAMPLES

General Experimental

Thin-layer chromatography was run on silica gel 60 F-254 (EM Reagents)aluminum-backed plates. ¹H NMR spectra were obtained at 300 MHz on aVarian VXR-300 spectrometer in DMSO-d₆. Elemental analyses wereperformed by Quantitative Technologies Inc. (Boundbrook, N.J.). Massspectrometry was performed by Mass Consortium (San Diego, Calif.). Allprocedures were carried out at room temperature unless otherwisespecified.

Example 1 Steady-State Fluorescence Measurements

Fluorescence spectra were recorded on a Perkin Elmer model MPF-44A, or aPerkin Elmer model LS50B fluorescence spectrophotometer at ambienttemperature. A Xenon lamp was used as the radiation source employing anexcitation wavelength appropriate for a particular dye (e.g., 485-507 nmfor thiazole orange).

For the experiments shown in FIGS. 2 and 3, concentrations of thiazoleorange conjugates were typically varied in the range of 3×10⁻⁸ M to5×10⁻⁷ M in pH 7.2 buffer (10 mM sodium cacodylate, 0.2 M NaCl, 1 mMEDTA), by serial dilution of a 5×10⁻⁷ M solution. For duplexmeasurements, an equal molar ratio of target sequence was added to a5×10⁻⁷ M solution of conjugate, followed by a 15 min incubation at 25°C. Serial dilutions were then performed as described above.

Fluorescence spectra of conjugates containing an environment-sensitivefluorophore were typically taken at a concentration of 1×10⁻⁷ M in pH7.4 buffer (10 mM phosphate, 0.15 M NaCl, 1 mM EDTA). Hybrids wereformed by adding 1-2 equivalents of target sequence.

Example 2 Synthesis of Oligonucleotides (ODNs)

All ODNs were prepared from 1 μmol appropriate CPG support on an ABI 394synthesizer using the protocol supplied by the manufacturer. Protectedβ-cyanoethyl phosphoramidites of 2′-deoxyribo and2′-O-methylribonucleotides, CPG supports, deblocking solutions, capreagents, oxidizing solutions and tetrazole solutions were purchasedfrom Glen Research. 5′-Aminohexyl modifications were introduced using anN-(4-monomethoxytrityl)-6-amino-1-hexanol phosphoramidite linker (GlenResearch). 3′-Aminohexyl and 3′-hexanol modifications were introducedusing a modified CPG prepared as previously described. Petrie et al(1992) Bioconjugate Chem. 3:85-87; and U.S. Pat. No. 5,212,667. Allother general methods employed for preparative HPLC purification,detritylation and butanol precipitation were carried out as described.Reed et al. (1991) Bioconjugate Chem. 2:217-225. Purifiedoligonucleotides were analyzed by C-18 HPLC (column 250×4.6 mm) in agradient of 0-30% acetonitrile in 0.1 M triethylamine acetate buffer, pH7.0, over 20 min at a flow rate of 2 ml/min. Pump control and dataprocessing were performed using a Rainin Dynamax chromatographicsoftware package on a Macintosh computer. ODN purity was assessed bycapillary gel electrophoresis (CGE) with a P/ACE™ 2000 Series equippedwith an eCAP™ cartridge (Beckman, Fullerton, Calif.). Oligonucleotideswere >95% pure by C-18 HPLC and showed one major peak on CGE.

Example 3 Synthesis of p-nitrophenyl Carbonate-Activated LatentFluorophores

1-(3-Hydroxypropyl)-4-methylquinolinium bromide (3). A solution oflepidine (0.49 g, 3.43 mmol) and 3-bromo-1-propanol (3.1 ml, 34 mmol) in3.0 ml of 1,4-dioxane was refluxed for 17 h. The solution was cooled toroom temperature and then diluted with 30 ml of ether. The productseparated as an oil and the ether layer was discarded. The oil wascrystallized from methylene chloride: 367-mg (38%) yield; TLC (5:3:2,n-butanol/water/acetic acid), R_(f)=0.40; ¹H NMR δ 9.39 (1H, d, J=6.0Hz, aromatic), 8.57 (2H, t, J=9.1 Hz, aromatic), 8.27 (1H, t, J=7.8 Hz,aromatic), 8.05 (2H, m, aromatic), 5.08 (2H, t, J=7.1 Hz, methylene),4.81 (1H, t, J=4.9 Hz, hydroxyl), 3.51 (2H, m, methylene), 3.01 (3H, s,4-methyl), 2.11 (2H, m, methylene). Anal. Calcd. For C₁₃H₁₆BrNO 0.3H₂O;C, 54.29; H, 5.82; N, 4.87. Found C, 53.92; H, 5.43; N, 4.67.

1-(3-Hydroxypropyl)-thiazole orange (4). To a solution of3-methyl-2-thiomethyl-benzothiazolium iodide (0.38 g, 1.22 mmol) and 3(0.34 g, 1.22 mmol) in 40 ml of absolute ethanol was added triethylamine(0.26 ml). The solution was stirred for 30 minutes at room temperatureand the crystals that formed were filtered, rinsed with ethanol anddried: 283 mg. yield; TLC (5:3:2, n-butanol/water/acetic acid),R_(f)=052; ¹H NMR δ 8.81 (1H, d, J=8.3 Hz), 8.61 (1H, d, J=7.4 Hz), 8.14(1H, d, J=8.6 Hz), 8.02 (2H, m), 7.77 (2H, m), 7.61 (1H, t, J=7.4 Hz),7.40 (2H, m), 6.93 (1H, s), 4.82 (1H, t, J=4.7H, hydroxyl), 4.66 (2H, t,J=6.5 Hz, methylene), 4.02 (3H, s, methyl), 3.50 (2H, m, methylene),2.01 (2H, m, methylene). Anal. Calcd. For C₂₁H₂₁IN₂OS.0.95H₂O; C, 51.11;H, 4.68; N, 5.68. Found C, 50.76; H, 4.23; N, 5.42.

4-Nitrophenyl carbonate derivative (5). 4-Nitrophenyl chloroformate (48mg, 0.240 mmol) and 4 (50 mg, 0.120 mmol) were stirred in 6.0 ml ofanhydrous pyridine at 70° C. for 2 h. Another portion of 4-nitrophenylchloroformate (48 mg) was added and stirring was continued for anotherhour. The solution was evaporated to dryness and the residue wascrystallized from DMF-THF. The red solid was filtered, rinsed with THFand dried: 29 mg yield; TLC (5:3:2, n-butanol/water/acetic acid),R_(f)=0.58; ¹H NMR δ 8.81 (1H, d, J=8.5 Hz), 8.60 (1H, d, J=7.4 Hz),8.27-7.97 (5H, m), 7.77 (2H, m), 7.62 (1H, t, J=7.4 Hz), 7.50-7.32 (4H,m), 6.93 (1H, s), 4.70 (2H, t, J=6.8 Hz, methylene), 4.03 (3H, s,methyl), 3.79 (2H, t, J=6.0 Hz, methylene), 2.33 (2H, m, methylene).HRMS (FAB) m/e 514.1416 M⁺, calcd for C₂₈H₂₄N₃O₅S, 514.1437.

Example 4 Synthesis of 2,3,5,6-tetrafluorophenyl3-[(3-{[3-(6-{[(4-methoxyphenyl)diphenylmethyl]amino}hexanoyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indolin-7-yl)carbonyl]pyrrolo[4,5-e]indoline-7-carboxylate(12) according to Reactions Scheme 4

6-{([(4-methoxyphenyl)diphenylmethyl]amino}hexanoic acid,triethylammonium salt (6). A suspension of 6-aminohexanoic acid (5.0 g,38 mmol) in 50 ml of anhydrous pyridine was treated withp-anisylchlorodiphenylmethane-MMTrCl (6.0 g, 19.4 mmol). After beingstirred for 24 hours at room temperature, the mixture was concentrated,and the residue, a viscous liquid, was partitioned between water andCH₂Cl₂. The organic layer was washed with water and dried over anhydroussodium sulfate. The crude product was chromatographed on silica elutingwith 5% MeOH, 0.5% triethylamine in CH₂Cl₂. Concentration of the properfractions and drying under vacuum afforded 2.2 g (22% yield) of thedesired MMTr-derivative as a pale-yellow, viscous oil.

2,3,5,6-tetrafluorophenyl6-{[(4-methoxyphenyl)diphenylmethyl]amino}hexanoate (7). The acid 6obtained as described above (2.2 g, 4.4 mmol) was dissolved in anhydrousCH₂Cl₂ and treated with 1 ml of triethylamine followed by 0.8 ml of2,3,5,6-tetrafluorophenyltrifluoroacetate. After being kept at roomtemperature for 30 min, the reaction was concentrated to an oil (crude7), which then was re-suspended in 20% ethyl acetate/80% hexane andapplied to a silica gel column. Elution with 15% ethyl acetate/85%hexane and concentration of the pure product fractions afforded 2.0 g(82%) of the TFP ester (7) as a colorless, viscous oil.

Methyl3-(6-{[1-(4-methoxyphenyl)-2-methylene-1-phenylbut-3-enyl]amino}hexanoyl)pyrrolo[4,5-e]indoline-7-carboxylate(8). A solution of 7 (0.6 g, 1.1 mmol) was combined with 0.24 g (1.2mmol) methylpyrrolo[4,5-e]indoline-7-carboxylate (Boger et al, supra)and 0.1 ml triethylamine in 5 ml of anhydrous CH₂Cl₂. The mixture waskept at room temperature for 15 h and concentrated under vacuum. Theresultant solid, which was the desired product, was washed with 50%ethyl acetate/50% hexane to remove unreacted starting materials and2,3,5,6-tetrafluorophenol. Drying under vacuum afforded 0.51 g (77%) ofthe title compound as a pale-yellow, crystalline solid.

3-(6-{[1-(4-methoxyphenyl)-2-methylene-1-phenylbut-3-enyl]amino}hexanoyl)pyrrolo[4,5-e]indoline-7-carboxylicacid (9). A mixture of 8 (0.47 g, 0.78 mmol), THF (9 ml), MeOH (6 ml)and 4M LiOH (3 ml) was stirred at 55° C. for 1 h. The resultant solutionwas cooled to give a white precipitate, the Li salt of the product. Thesolid was triturated with a small amount of cold 10% citric acid andfiltered off. Washing with water and drying under vacuum gave 0.43 g(94%) of 9 as a white solid.

Methyl3-[(3-{[3-(6-{[(2E)-1-(4-methoxyphenyl)-2-methyl-1-phenylpenta-2,4-dienyl]amino}hexanoyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indolin-7-yl)carbonyl]pyrrolo[4,5-e]indoline-7-carboxylate(10). To a solution of 9 (213 mg, 0.36 mmol) and methyl3-(pyrrolo[4,5-e]indolin-7-ylcarbonyl)pyrrolo[4,5-e]indoline-7-carboxylate(which had been prepared by TFA deprotection of 182 mg of thecorresponding t-Boc precursor, Boger et al., supra) in 50 ml ofanhydrous DMF was added EDC (200 mg). The reaction was stirred for 20 h25° C. The resultant precipitate was collected by filtration, thenwashed with MeOH and ether. Drying under vacuum afforded 313 mg (90%) ofthe desired product as an off-white solid.

3-[(3-{[3-(6-{[(2E)-1-(4-methoxyphenyl)-2-methyl-1-phenylpenta-2,4-dienyl]amino}hexanoyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indolin-7-yl)carbonyl]pyrrolo[4,5-e]indoline-7-carboxylicacid (11). A suspension of 10 (270 mg, 0.28 mmol) in a mixture of THF (6ml), MeOH (4 ml) and 4M LiOH (2 ml) was stirred at 55° C. for 30 h. Thereaction was cooled and neutralized to pH 6 with cold 10% citric acid.Insoluble material was collected by filtration and washed with water,MeOH and ether. Drying under vacuum afforded 160 mg (60%) of the titlecompound 11. By HPLC analysis this product contained ˜5% of unreacted10. The crude acid was used in the next step without additionalpurification.

2,3,5,6-tetrafluorophenyl3-[(3-{[3-(6-{[(4-methoxyphenyl)diphenylmethyl]amino}hexanoyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indolin-7-yl)carbonyl]pyrrolo[4,5-e]indoline-7-carboxylate(12). To a suspension of 11 (153 mg, 0.16 mmol) in 5 ml of anhydrous DMFwere added triethylamine (0.3 ml) and tetrafluorophenyl trifluoroacetate(TFP-TFA, 0.2 ml). The mixture was stirred for about 1 h at 25° C. togive an almost clear solution. The solution was filtered andconcentrated under vacuum to an oil. The oil was triturated withmethanol to produce a precipitate of the desired TFP ester 12. It wascollected by filtration, washed with MeOH then ether, and dried. Yieldwas 154 mg (90%). This product was ˜90% pure by HPLC analysis. Nofurther purification was attempted due to its poor solubility.

Example 5 Synthesis of an ODN-CDPI₃-thiazole orange conjugate (14)according to Reaction Scheme 5

5′-hexylamine modified 15-mer ODNs were prepared and the 5′-MMT groupwas removed on the synthesizer, using standard conditions. The5′-hexylamine modified ODN was reacted with the TFP activated 12, thendeprotected with aqueous TFA to yield the ODN-MGB conjugate 13. Thisconjugate was purified by reverse phase HPLC using triethylammoniumacetate/acetonitrile and the desired fraction was dried on a centrifugalevaporator (SpeedVac). The residue was dissolved in 20 μl of dry DMSO.To determine the concentration, 1 μl was removed and precipitated from2% sodium perchlorate. The pellet was washed with acetone, then driedand dissolved in water. Concentration of the initial DMSO solution wasdetermined by A₂₆₀ to be 1.68 mM, using a calculated extinctioncoefficient for the CDPI₃-amine-ODN conjugate of ε=255,000 M⁻¹cm⁻¹.

15 μl of the DMSO solution of the ODN-CDPI₃ conjugate (25.2 nmol) wastreated with 1 mg (2 μmol) of the 4-nitrophenyl carbonate derivative ofthiazole orange (5) and 5 μl of triethylamine. After shaking for 16 h atroom temperature, the crude conjugate was precipitated from 1 ml of 2%sodium perchlorate. The orange pellet was washed with acetone, dried ona SpeedVac and dissolved in 100 μl water. The conjugate (14) waspurified by reverse-phase HPLC using triethylammoniumacetate/acetonitrile, the fraction containing the conjugate wasconcentrated to 0.1 ml with butanol, and the conjugate was precipitatedwith 2% sodium perchlorate. The orange pellet was washed with acetone,dried on a SpeedVac, and dissolved in 50 μl water to give a 0.43 mMsolution. An absorbance spectrum showed distinctive absorbances due tothe ODN (260 μm), CDPI₃ (350 nm) and thiazole orange (500 nm).

Example 6 Synthesis of CPG-CDPI₃ derivative (23) according to ReactionScheme 7

4-nitrophenyl {2-[(2-hydroxyethyl)suyfonyl]ethoxy}formate (18). Asolution of 2,2′-sulfonyldiethanol (4.85 g, 39.75 mmol) andp-nitrophenyl chloroformate (2.0 g, 9.92 mmol), in 20 ml of drypyridine, was stirred for 2 h at room temperature and then evaporated todryness. The residue was dissolved in 350 ml of ethyl acetate and washedwith water (4×100 ml). The organic solution was dried over sodiumsulfate, filtered and evaporated. The crude product was purified bysilica gel chromatography, eluting with ethyl acetate. The pure productfractions were pooled and evaporated affording an oil: 0.68 g (22%)yield.

2-({2-[N-(3-{[(4-methoxyphenyl)diphenylmethyl]amino}propyl)carbamoyloxy]-ethyl}sulfonyl)ethyl(4-nitrophenoxy)formate (20). A solution of 18 (0.68 g, 2.13 mmol) and(3-aminopropyl)[(4-methoxyphenyl)diphenylmethyl]amine (0.89 g, 2.56mmol) was stirred at 40° C. for 30 min. p-nitrophenyl chloroformate(0.62 g, 3.08 mmol) was added and stirring was continued for anadditional 2 h. The solution was diluted with ethyl acetate (350 ml),washed with water (300 ml) and then dried over sodium sulfate andevaporated. The residue was purified by silica gel chromatographyeluting with a gradient of 40-100% ethyl acetate in hexane. The pureproduct fractions were evaporated affording a foam: 351 mg of 20 (35%)yield; ¹H NMR (DMSO-d₆) δ 8.32 (2H, d, J=9.2 Hz, aromatic), 7.56 (1H, d,J=9.2 Hz, aromatic), 7.37 (4H, d, J=7.4 Hz, aromatic), 7.29-7.15 (8H, m,aromatic), 6.83 (2H, d, J=8.9 Hz, aromatic), 4.61 (2H, t, J=5.5 Hz,CH₂), 4.29 (2H, t, J=6.0 Hz, CH₂), 3.71 (3H, s, methoxy), 3.67 (2H, t,J=5.7 Hz, CH₂), 3.51 (2H, t, J=5.8 Hz, CH₂), 3.07 (2H, m, CH₂), 1.93(2H, m, CH₂), 1.59 (2H, m, CH₂).

Synthesis of CPG derivative 21. A mixture of 20 (325 mg, 0.47 mmol) andlong chain alkyl amino CPG (5.9 g) was swirled in 24 ml of dry pyridinefor 20 h at 25° C. Acetic anhydride (20 ml) was added and the mixturewas swirled for an hour at 25° C. and then filtered. The glass beads 21were rinsed generously with dimethylformamide and ethyl acetate anddried under vacuum.

Synthesis of CPG-CDPI₂-derivative (22). A portion of beads 21 (1.5 g)was deprotected by suspending the beads in 3% trifluoroacetic acid inmethylene chloride for 5 min. and then filtered. This process wasrepeated twice. On the third filtration step the filtrate was no longercolored. The beads were rinsed with methylene chloride and then with 50ml of a solution of 5% triethylamine in acetonitrile, followed by rinseswith pure acetonitrile and then ether.

The deprotected beads were mixed with activated ester 24 (140 mg, 0.22mmol) in 6.0 ml of pyridine/DMF (1:1 v/v) and the mixture was swirledfor 18 h at room temperature. Activated ester 24 was prepared accordingto Lukhtanov et al. (1995) Bioconjugate Chemistry 6:418-426. Aceticanhydride (1.0 ml) was added and the mixture was swirled for 1 h at roomtemperature and then filtered. The product beads 22 were rinsed with DMFand ethyl acetate and dried under vacuum.

Synthesis of CPG-CDPI₃ derivative (23). A suspension of 22 in 15 ml oftrifluoroacetic acid was swirled for 1 h at room temperature and thenfiltered. The beads were rinsed with methylene chloride and then with 50ml of 10% triethylamine in acetonitrile followed by ethyl acetate. Thebeads were then dried under vacuum.

Activated ester 25 was prepared according to Lukhtanov et al (1997a)supra.

A mixture of the glass beads 22 and activated ester 25 (103 mg, 0.22mmol) was swirled in 6.0 ml of dry pyridine for 18 h at room temperatureand then treated with 3.0 ml of acetic anhydride. The mixture wasswirled for an additional hour at room temperature and then filtered.The product beads 23 were rinsed with DMF and ethyl acetate and thendried under vacuum.

Synthesis of CPG derivative (15) for oligonucleotide synthesis. Asuspension of 23 in 15 ml of trifluoroacetic acid was swirled for 1 h atroom temperature and then filtered. The beads were rinsed with methylenechloride and then with 50 ml of 10% triethylamine in acetonitrilefollowed by ethyl acetate. The beads were dried under vacuum.

A mixture of the beads and 4-nitrophenyl4-[bis(4-methoxyphenyl)phenyl-methoxy]butanoate (200 mg, 0.378 mmol) wasswirled in 6.0 ml of dry pyridine for 18 h at room temperature and thentreated with 3.0 ml of acetic anhydride. The mixture was swirled for anadditional hour at room temperature and then filtered. The product beads15 were rinsed with DMF and ethyl acetate and then dried under vacuum.Loading of the beads was 16.7 μmol/g.

Example 7 Synthesis of ODN-MGB-LF (11) in Reaction Scheme 6

The CPG-beads 15 prepared as in Example 6 were deprotected withTFA/CH₂Cl₂ and used for oligonucleotide synthesis under standardconditions. After synthesis of the oligonucleotide was complete, ammoniadeprotection yielded the aminopropyl-CDPI₃-ODN derivative 16. Reactionof 16 with a reactive fluorophore derivative (e.g., 5) yielded anODN-MGB-LF conjugate 17.

Example 8 Mismatch Discrimination using ODN-MGB-TO Conjugates

The ability of ODN-MGB-LF conjugates to discriminate between aperfectly-matched hybrid and a single-nucleotide mismatch was tested,using TO as the latent fluorophore portion of the conjugate.Discriminatory ability was expressed as ΔT_(max), the difference betweenthe T_(max) values for a perfect match and a single-nucleotide mismatch,where T_(max) is the temperature at which the rate of decrease influorescence (−dF/dt, indicative of denaturation of hybrid) is maximum.

ODN-MGB-TO conjugates with ODN portions ranging from 10-18 nucleotidesin length were hybridized, at a concentration of 1 μM, to an equimolarconcentration of either a target ODN containing a perfectly-matched(i.e., fully complementary) sequence or an ODN containing asingle-nucleotide mismatch. The perfectly-matched target had thesequence 5′-CTT CTT TTC TTT AAA TTG CC-3′ (SEQ ID NO: 8). The mismatchedtarget had the sequence 5′-CTT CTT TTC TTT CAA TTG CC-3′ (SEQ ID NO: 9).The position at which the mismatch occurs in the mismatchedoligonucleotide is underlined in all oligonucleotide sequences.Hybridization was conducted in 200 mM NaCl, 10 mM Na cacodylate, 1 mMEDTA, pH 7.2. The hybridization reactions were initially incubated for15 minutes at room temperature; then the temperature was increased to95° C. at a rate of 0.2° C. per second.

Fluorescence measurements were conducted on 7 μl of each hybrid, in anIdaho Technologies LC-24 Light Cycler according to the manufacturer'sinstructions. Fluorescence was continuously monitored at 560 nm and theresults are shown in Table 3.

TABLE 3 Mismatch discrimination using ODN-MGB-LF conjugates SEQ T_(max)of T_(max) of ODN-MGB-LF Conjugate ID NO Length match mismatch ΔT_(max)5′-TO-MGB-CAATTTAAAGAAAAGAAG 10 18 65° C. 55° C. 10° C.5′-TO-MGB-CAATTTAAAGAAAAGA 11 16 61° C. 48.5° C.  12.5° C. 5′-TO-MGB-CAATTTAAAGAAAA 12 14 58° C. 42° C. 16° C.5′-TO-MGB-CAATTTAAAGA 13 12 54° C. 35° C. 19° C. 5′-TO-MGB-CAATTTAAAG 1410 48° C. * * - duplexes not detected

These results indicate that ODN-MGB-LF conjugates are able todiscriminate between a perfectly-matched hybrid and a hybrid containinga single-nucleotide mismatch. Discrimination is achieved for sequencesas short as 10 nucleotides.

Example 9 ODN-MGB-Fluorophore Conjugates as Primers in Real-Time PCR

This example demonstrates that ODN-MGB-LF conjugates are useful asprimers in real-time PCR assays, and that single-nucleotide mismatchdiscrimination can be achieved in real-time PCR using ODN-MGB-LFconjugates. See Wittwer et al. (1997) supra for a description ofreal-time PCR.

Real-time PCR with fluorescent monitoring was performed in an IdahoTechnologies LC-24 Light Cycler. Each reaction mixture contained: 40 mMNaCl, 20 mM Tris-HCl, 5 mM MgCl₂, 0.05% bovine serum albumin, 125 μMeach dNTP, 0.5 μM each primer (including fluorescent primer), 0.1 ng/10μL template and 0.5 U/10 μL Taq Polymerase. Cycling conditions for thisexperiment were 40-50 cycles of 1 sec at 95° C., then 30 sec at theannealing/extension temperature of 71° C.

The template was the 4518 bp pBK-CMV phagemid (Stratagene; Alting-Mees,et al. (1992) Strategies 5:58-61. The template contained a LacZ geneinsert (ATG at position 1183, TAA at 799) in which the region betweennucleotides 1060 and 1083 was substituted with either the matched targetsequence 5′-TCT TTC TTC TTT TCT TTA AAT TGC CC-3′ (SEQ ID NO: 15) or themismatched target sequence 5′-TCT TTC TTC TTT TCT TTC AAT-3′ (SEQ ID NO:16).

The following primers were chosen to produce a 42 bp amplicon. Theforward primer was 5′-AACCCGCGGCCGCTCTA-3′ (SEQ ID NO: 17). Two reverseprimers, both containing a LF, were used. The first, which alsocontained a MGB, was 5′-TO-MGB-CAATTTAAAGAAAAGAAG-3′ (SEQ ID NO: 18).The second, which lacked a MGB, was 5′-TO-CAATTTAAAGAAAAGAAG-3′ (SEQ IDNO: 19).

FIG. 6 shows fluorescence as a function of cycle number for theODN-MGB-TO conjugate used as a PCR primer for a perfectly-matched vs. asingle-base mismatched primer binding sequence. A strong fluorescenceoutput is observed for the template with the perfectly-matched sequence;however, only background fluorescence is observed for the template withthe single-base mismatch. FIG. 6 also shows that a TO-conjugated,perfectly-matched primer lacking a MGB yields only backgroundfluorescence in this assay, confirming the beneficial effect of a MGBmoiety on hybridization-triggered fluorescence.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatvarious changes and modifications can be practiced without departingfrom the spirit of the invention. Therefore the foregoing descriptionsand examples should not be construed as limiting the scope of theinvention.

1. A method for detecting fluorescence in a cyanine dye having a minorgroove binder attached thereto, said method comprising: providing acyanine dye having a minor groove binder attached thereto, which isessentially nonfluorescent in the absence of double stranded nucleicacid; and contacting said cyanine dye with double stranded nucleic acidto trigger hybridization fluorescence.
 2. The method of claim 1, whereinintercalation of said cyanine dye having a minor groove binder intodouble stranded nucleic acid prevents free rotation of said cyanine dye.3. The method of claim 1, wherein when said cyanine dye is free insolution, free bond rotation allows the cyanine dye to transit from theexcited singlet state (S₁) to the ground state (S₀) in a radiationlessprocess.
 4. The method of claim 1, wherein said cyanine dye has anelectron donating group and an electron accepting group which arecovalently joined to each other by a resonance linker.
 5. The method ofclaim 1, wherein said minor grove binder further comprises anoligonucleotide, said oligonucleotide having a 3′ end and a 5′ end. 6.The method of claim 5, wherein said minor groove binder moiety iscovalently linked to the 5′ end of the oligonucleotide.
 7. The method ofclaim 5, wherein the minor groove binder moiety is covalently linked tothe 3′ end of the oligonucleotide.